Production and use of rat spermatogonial stem cell lines

ABSTRACT

A spermatogonial stem cell line that is derived from testes of rats characterized by a desirable genetic background can serve as a source for cells to transplant into male-sterile recipient animals that are immuno-compatible with the spermatogonial line. Rat cells thus transplanted readily develop into fertilization-competent, haploid male gametes, with little or no endogenous sperm competition generated by the testes of the male-sterile recipient. This approach, constituting the first vector system for the use of rat spermatogonial lines from in vitro culture in generating mutant rats on a desired genetic background, effects maximal germline transmission of donor haplotypes from the transplanted spermatogonial cells.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Number5R21RR023958-02 awarded by the National Institute of Health/NationalCenter for Research Resources (NIH/NCRR). The U.S. Government hascertain rights in the invention.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is the U.S. National Stage filing of InternationalApplication Serial No. PCT/US2009/066275, filed Dec. 1, 2009, whichclaims the benefit of U.S. Provisional Application No. 61/119,005, filedDec. 1, 2008 and U.S. Provisional Application No. 61/187,498, filed Jun.16, 2009, each of which are herein incorporated by reference in theirentireties.

BACKGROUND OF THE INVENTION

The present invention relates generally to the stem cell field and, moreparticularly, to employing stem cells in the development of rat celllines and animals.

Propagation of Spermatogonial Stem Cell Lines

The ability to conditionally induce the development of stem cell linesthrough the process of spermatogenesis in vitro for the production ofgametes would provide a long-sought-after technology for biomedicalresearch, particularly if such protocols could be established for avariety of species. The discovery that stem cells residing withinfractions of dissociated mouse and rat testis cells maintain theirability to regenerate spermatogenesis in testes of recipient mice wasessential to establishing such culture systems. See Brinster et al.,Proc Natl Acad Sci USA 1994; 91:11303-11307; Brinster et al., Proc NatlAcad Sci USA 1994; 91:11298-11302; Clouthier et al., Nature 1996;381:418-421; Kanatsu-Shinohara et al., Biol Reprod 2003; 69:612-616; andNagano et al., Tissue Cell 1998; 30:389-397. The ability to isolate andexperimentally manipulate these stem cells has opened new doors forresearch on spermatozoan development, assisted reproduction, cellulartherapy and genetics. See Nagano et al., Biol Reprod 1999; 60:1429-1436;Mahato et al., Endocrinology 2000; 141:1273-1276; Mahato et al., MolCell Endocrinol 2001; 178:57-63; Ogawa et al., Nat Med 2000; 6:29-34;Shinohara et al., Proc Natl Acad Sci USA 2006; 103:13624-13628; Zhang etal., J Cell Physiol 2007; 211:149-158; Kazuki et al., Gene Ther 2008;15:617-624; Kanatsu-Shinohara et al., Cell 2004; 119:1001-1012;Kanatsu-Shinohara et al., Proc Natl Acad Sci USA 2006; 103:8018-8023;and Nagano et al., Proc Natl Acad Sci USA 2001; 98:13090-13095. In viewof this potential, protocols for isolating, propagating and geneticallymodifying fully functional rat spermatogonial stem cells in culture havebeen established. See Ryu et al., Dev Biol 2004; 274:158-170; Hamra etal., Dev Biol 2004; 269:393-410; Hamra et al., Proc Natl Acad Sci USA2002; 99:14931-14936; Hamra et al., Methods Mol Biol 2008; 450:163-179;Hamra et al., Proc Natl Acad Sci USA 2005; 102:17430-17435; Ryu et al.,Proc Natl Acad Sci USA 2005; 102:14302-14307; Orwig et al., Biol Reprod2002; 67:874-879; and Kanatsu-Shinohara et al., Biol Reprod 2008. Therat was chosen as a species for these studies due to its popularity as alaboratory animal model for the study of human health and disease, anddue to the lack of protocols for genetically modifying the rat germlineusing clonally expanded stem cells from culture. See Hamra et al., ProcNatl Acad Sci USA 2002; 99:14931-14936 Considering the many potentialapplications of the laboratory rat as a research model, a cost-effectiveand easy-to-prepare culture medium was sought in this study for thederivation and continuous proliferation of primary rat spermatogonialstem cell lines in vitro.

In respect of this goal, media previously reported for long-termproliferation of rodent spermatogonial stem cells in vitro representclear methodological advances for studies on the biology andapplications of spermatogonia. See Kanatsu-Shinohara et al., Biol Reprod2003; 69:612-616; Hamra et al., Proc Natl Acad Sci USA 2005; Ryu et al.,Proc Natl Acad Sci USA 2005; 102:14302-14307; Kubota et al., Proc NatlAcad Sci USA 2004; 101:16489-16494; and Kanatsu-Shinohara et al., BiolReprod 2005; 72:985-991. However, such media are relatively complex,expensive, time-consuming to prepare, plus are most effective whenapplied in combination with feeder layers of fibroblasts. See id. Forexample, the medium originally reported by Shinohara and colleagues forthe successful derivation and long-term cultivation of germline stemcells from postnatal mouse testes was a pivotal breakthrough inspermatogonial research. See Kanatsu-Shinohara et al., Biol Reprod 2003;69:612-616. However, Shinohara's medium is based on the proprietary,StemPro-34 medium, plus 24 individually added components, includingsmall molecules, fetal bovine serum and a mixture of polypeptide growthfactors. Serum-free derivatives of Shinohara's medium have since beenformulated for spermatogonial culture, in which the serum has beenreplaced by the supplement, B-27. See Hamra et al., Proc Natl Acad SciUSA 2005; 102:17430-17435 and Kanatsu-Shinohara et al., Biol Reprod2005; 72:985-991. Upon inspection of components within B-27 supplementwe postulated that it could be used together with key growth factors ina commonly applied nutrient mixture to formulate a more efficientspermatogonial culture medium.

Sterile Testes Complementation

Currently, specific causes of infertility in men remain a mystery in40-60% of cases. See Bhasin et al., J Clin Endocrinol Metab 79, 1525-9(1994); Sadeghi-Nejad et al., Urol J 4, 192-206 (2007); and Matzuk etal., Nat Med 14, 1197-213 (2008). In total, >5% of the male populationis infertile, and >1% of all males are inflicted with a severe defect insperm production termed azoospermia. See Bhasin et al., J ClinEndocrinol Metab 79, 1525-9 (1994); Sadeghi-Nejad et al., Urol J 4,192-206 (2007); Barthold et al. J Urol 170, 2396-401 (2003); and Bleyer,W. A. CA Cancer J Clin 40, 355-67 (1990). Fundamentally, becauseazoospermia results in an inability to reproduce by natural mating, itseems enigmatic as to why this disease remains so prevalent in the humanpopulation. Such an epidemiological trend clearly points to theexistence of potent environmental factors that disrupt the process ofsperm production (i.e. spermatogenesis) or a substantial number of denovo mutations that could arise during a lifetime to render one sterile,but otherwise healthy. See Bhasin et al., J Clin Endocrinol Metab 79,1525-9 (1994); Bleyer, W. A., CA Cancer J Clin 40, 355-67 (1990); Reijo,R. et al. Nat Genet. 10, 383-93 (1995); Oates et al., Hum Reprod 17,2813-24 (2002). In fact, this is true in numerous cases, as such de novomutations account for several types of male-factor infertility alreadydefined at a genetic level and increasing numbers of males are leftinfertile during their childhood by cancer chemotherapy. SeeSadeghi-Nejad, et al., Urol J 4, 192-206 (2007); Reijo et al. Nat Genet.10, 383-93 (1995); Bleyer et al., CA Cancer J Clin 40, 355-67 (1990);Oates et al., Hum Reprod 17, 2813-24 (2002); Bhasin, S., J ClinEndocrinol Metab 92, 1995-2004 (2007); and Geens, M. et al., Hum ReprodUpdate 14, 121-30 (2008). As a new hope for many infertile men withazoospermia, a pioneering breakthrough in stem cell biology thatmanifested strong links between reproductive biology and geneticresearch was the discovery that mouse testes contained spermatogonialstem cells capable of generating fully functional sperm followingisolation and transplantation into testes of another mouse. See Brinster& Zimmermann, Proc Natl Acad Sci USA 91, 11298-302 (1994). Similarexperiments soon followed in rats, and isolated mouse spermatogonia werenext shown to maintain their regenerative potential after months inculture. See Clouthier et al., Nature 381, 418-21 (1996); Nagano et al.,Tissue Cell 30, 389-97 (1998). New culture media supporting the longterm proliferation of rodent spermatogonial lines in vitro have sincebeen formulated and scientists are now on the brink of establishingconditions required to cultivate human spermatogonial lines from testisbiopsies. See Kanatsu-Shinohara et al., Biol Reprod 69, 612-6 (2003);Hamra, F. K. et al., Proc Natl Acad Sci USA 102, 17430-5 (2005); Conrad,S. et al. Nature (2008); and Kossack, N. et al. “Isolation andCharacterization of Pluripotent Human Spermatogonial Stem Cell-DerivedCells.” Stem Cells (2008). Ostensibly, the ability to propagatespermatogonial lines in culture, prior to using them to producefunctional spermatozoa by transplanting them back into the testes oftheir own donor, presents a clear strategy to cure many existing typesof male infertility. Due in large part to the multipotent nature ofgermline stem cells however, before these breakthroughs are translatedinto practice it is imperative that preclinical details of such cellulartherapies first be stringently evaluated in more advanced, non-humanrecipients of medical relevance. See Geens, M. et al., Hum Reprod Update14, 121-30 (2008); Conrad, S. et al. “Generation of pluripotent stemcells from adult human testis.” Nature (2008); Kossack, N. et al.“Isolation and Characterization of Pluripotent Human Spermatogonial StemCell-Derived Cells.” Stem Cells (2008); Hermann, B. P. et al. Stem Cells25, 2330-8 (2007); and Zhang et al., J Cell Physiol 211, 149-58 (2007).

Production of Transgenic Animals

In mice, embryonic stem (ES) cell-based knockout technology is veryefficient for single gene targeting, and it can be combined as well withthe usage of random mutagens, such as chemical mutagenic agents, virusesor transposons, for the large-scale generation of ES cell libraries,carrying different molecularly marked knockout alleles. These ES cellclones can be used for the production of knockout mice.

While this methodology is applicable for mice, it cannot be employedwith rats or with other laboratory animals. Furthermore, no similar orequivalent techniques to the mouse ES cell technique have yet beendeveloped that would be applicable to a variety of animal models and notlimited to one animal species like the ES cell technique in mice. Forexample, due to the above-mentioned technical limitation very few ratknockout strains are currently existing worldwide. This may at leastpartially be the result of the practicability of random mutagenesis inanimals, which has proven to be questionable for several reasons. Forexample, the requirement of a large number of offspring, the time forrearing offspring, the costs of establishing and maintaining large-scaleanimal facilities are some of the factors to be considered whengenerating transgenic animals using random mutagenesis in animals.Accordingly, there exists a need for more advantageous methods oftargeted mutagenesis that can be applied in a variety of animal modelsand are more practicable.

Current technologies used to create transgenic rats require a high levelof expertise and are costly to produce. Additionally, there are manydisadvantages to the currently available recipient rat models fortesticular transplantation of donor stem cells. These disadvantagesinclude but are not limited to: (1) lower germline transmission from thedonor cells to progeny, due to high levels of competition fromendogenous sperm cell production; (2) the need for a high number ofstems cells to be transplanted into recipient testes to producetransgenic progeny; (3) a large number of progeny must be produced toyield the desired mutant rat line; and (4) the need for a high dose ofcytotoxic chemicals or irradiation to achieve effective engraftment oftestes by donor stem cells. In conventional protocols, moreover, themost effective levels of stem cell engraftment have not been realizedbecause lethal doses of irradiation or cytotoxic reagents required foreffective stem cell engraftment kill the recipients. Finally, productionof rat lines with loss or gain of function gene mutations requirestedious, time-consuming and prohibitively expensive micromanipulation ofembryos.

There is a general need to annotate the human genome with function,linking laboratory animals into this process is a necessary requirementfor accelerating improvements in health care. For example, extensivephenotyping and detailed analysis of inbred animals strains has resultedin the localization of hundreds of loci involved in complex diseases.These “quantitative trait loci” (QTLs) demonstrate genetic linkage tomany disease traits which are shared between laboratory animals, such asrats, and humans. Examples for such diseases include hypertension,neuronal regeneration, ischemic cerebrovascular and cardiovasculardiseases, and diabetes. See Jacob, H. J. et al., Nature Rev. Genet. 3:33(2002), and Hubner, N. et al., loc. cit. 37:243 (2005).

SUMMARY OF THE INVENTION

The claimed invention comprehends, in part, using certain naturallyoccurring or transgenically generated (“genetically”) male-sterile ratsas recipients for donor sperm stem cells with which the rats also areimmuno-compatible. Spermatogenesis in these rats is severely disrupted,but they maintain a functional stem cell compartment. Accordingly, thetransplanted sperm stem cells are free to develop into functionalspermatozoa and to fertilize female rats in the absence of competitionfrom sperm that also would be produced, were the recipientsmale-fertile. In this manner, 100% germline transmission of the donorcell haplotype can be achieved from a relatively low number oftransplanted sperm stem cells.

Thus, pursuant to one aspect of the present invention, a methodology isprovided for effecting germline transmission of a rat donor haplotype.The inventive method comprises the steps of (A) providing cells of aspermatogonial stem cell line that is derived from rat testes, whichcell line embodies a predetermined genetic background, and then (B)transplanting one or more of the cells into a male-sterile recipient ratthat can be, for example, the product of crossing a DAZL-deficienttransgenic rat into the aforementioned genetic background, the recipientrat being immuno-compatible with the cells, such that transplanted cellsdevelop into fertilization-competent, haploid male gametes.

In accordance with another aspect of the invention, a library isprovided of cells of a spermatogonial stem cell line that is derivedfrom rat testes. A library of the invention contains a plurality oftransposon-mediated gene knockout or “knockin” mutant stem cells.

In accordance with an additional aspect of the invention, a medium forgrowing spermatogonial stem cells is provided, in addition to methodsfor culturing spermatogonial stem cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. DAZL-Deficient Rat Testes are Effectively Colonized by DonorSpermatogonia. (A) Relative abundance of EGFP in testes of Wildtype,DAZL-deficient and GCS-EGFP rats. Left: Data expressed as theequivalents of recombinant, histidine-tagged EGFP (rEGFP)/testis(+/−SEM, n=3 testes/rat strain) as determined by fluorometry of testisextracts at 24 days of age. Right: Bright field (top) and greenfluorescence (bottom) images of testes dissected from wildtype (WT),DAZL-deficient (Dazl-Def) and GCS-EGFP rats at 24 days of age. Scalebar=1 cm. (B) Spermatogenesis colony forming assays using DAZL-deficientrats as recipients. Left: Numbers of spermatogenic coloniesformed/testis by donor GCS-EGFP rat spermatogonia in Wildtype(10.25+/−0.68 colonies/testis, +/−SEM, n=8 testes) and DAZL-Deficientrats (30.69+/−0.62 colonies/testes, +/−SEM, n=8 testes) at 30 daysfollowing transplantation. Donor spermatogonia were transplanted atpassages 15 and 17 (i.e. culture days 182 and 204) at 2000 GCS-EGFP⁺cells/testis. Right: Images of individual colonies of spermatogenesis inWildtype and DAZL-deficient recipient rats that were generated by thedonor GCS-EGFP spermatogonia (green fluorescence is from donor cells).Images are representative of colonies scored and plotted in the Leftpanel. Scale bar=100 μm.

FIG. 2. Maximal Germline Transmission from DAZL-Deficient Rats. (A)Southern blot analysis of progeny from Wildtype (WT) and DAZL-Deficient(Dazl) recipient rats with right testes transplanted with 50,000GCS-EGFP spermatogonia at passage 13 (i.e., 158 days in culture); theleft testis of each animal was not transplanted. At 75 dayspost-transplantation recipients (R) were paired with WT females (F) andallowed to produce pups by natural breeding (See R942-R949 in Table 1).Shown are blots from representative litters probed for EGFP todistinguish progeny produced by donor cells from their recipient-fathersand wildtype mothers. OMP=loading control probe for genomic DNA onblots. Genomic DNA samples used for Controls on each blot were fromuntreated GCS-EGFP and DAZL-Deficient transgenic rats. LTR=PCR primersspecific for detecting the lentiviral transgene used to makeDAZL-deficient rats. GAPDH=PCR primers for genomic DNA loading control.(B) Bright field and green fluorescence images of testes from Wildtype(Left) and DAZL-deficient (Right) recipient rats at 212 dayspost-transplantation. Scale bar=1 cm. (C) Graph of germline transmissionrates by natural breeding for the donor GCS-EGFP transgene from Wildtypeand DAZL-deficient recipient rats that had their right testistransplanted with 50,000 GCS-EGFP spermatogonia at passage 13; the lefttestis of each animal was not transplanted. DAZL-deficient recipientstransmitted the GCS-EGFP transgene to 100%+/−0% of progeny (+/−SEM, n=3recipients; 9 litters), with 73 of 73 total F1 pups born from donorcells. Wildtype recipient littermates transmitted the GCS-EGFP transgeneto 14%+/−5.9% of progeny (+/−SEM, n=3 recipients; 9 litters), with 16 of116 total F1 pups born from donor cells. (D) Genealogy tree showingstable transmission of donor cell haplotypes from DAZL-Deficientrecipient/founders (F0) R989 and R990 to F1 and F2 progeny. Recipientswere each transplanted with 150,000 rat spermatogonia/testis from lineRSGL-GCS9 at passage 17 (See R988-R990 in Table 1). Spermatogonial lineRSGL-GCS9 was derived from a rat homozygous for the GCS-EGFP transgene.Thus, F1 progeny represent half-siblings; some of which were crossed tore-derive transgenic F2 progeny homozygous for the tgGCS-EGFP allele.

FIG. 3. Long-Term Spermatogenesis Colonizing Potential of DonorSpermatogonia (Left) Graph showing relative numbers of Round andElongating Spermatids in seminiferous tubules of non-transplanted,non-busulfan-treated, Wildtype and DAZL-deficient rat lines at 4 monthsof age (see FIG. 1 a), in comparison to Spermatid numbers inbusulfan-treated, DAZL-deficient recipient rats at 212 days (i.e. ˜8months of age) after being transplanted with rat spermatogonial line,RSGL-GCS9, at passage 13 (i.e. culture day 158). Cell counts werenormalized/1000 Sertoli cells. +/−SEM, n=3 rats/group. (Right) Images ofhistological sections of seminiferous tubules from the DAZL-deficientrecipient rats described in the “Left” panel after being transplantedwith spermatogonia from RSGL-GCS9. Bottom Right shows a highermagnification image within the boxed region of the Top Right panel.Scale bars=100 μm.

Table 1. Progeny from Wildtype and DAZL-Deficient recipient ratstransplanted with GCS-EGFP rat spermatognia

Table 2. Components of Rat Spermatogonial Culture Media

Table 3. Vendor and Catalog numbers for SA and SG media components

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Inventors have achieved virtually 100% germline transmission to ratprogeny by mating from donor primary lines of rat spermatogonial stemcells, following their propagation in culture, cryopreservation inliquid nitrogen, thawing, further expansion in culture, transplantationinto the testes of recipient, male-sterile rats, and development intofunctional spermatozoa.

Rat spermatogonial stem cells are rat cell types that when transplantedinto the testes can develop into fertilization competent haploidgametes, such as spermatids or spermatozoa. Natural mating or breedingof the recipient with a female rat bypasses any requirement for assistedfertilization methods. Additionally, mating can include methods such asbut not limited to in vitro fertilization, intrauterine transfer, oroocyte injection with nuclei. Sperm produced from the spermatogoniallines in the recipient rats are highly effective at transmitting thedonor haplotype to F1 and F2 progeny by mating, thereby renderingunnecessary conventional embryo manipulations to generate rat progenyfrom donor spermatogonial lines.

Pursuant to the invention, therefore, the combination of (i) ratspermatogonial lines that can be propagated in culture and (ii)male-sterile recipient rats is an optimal vector for germlinetransmission of natural or genetically modified rat genomes. Thisapproach will allow for preservation of existing rat lines and theproduction of new transgenic rat lines important for science andindustry. For example, the invention offers avenues to the establishmentof new transgenic mutant rat models, for the study of human biology anddisease.

In accordance with the invention, spermatogonial stem cell lines arederived from testes of rats, such as inbred Fischer 344 rats, thatembody a desirable genetic background. The derived lines are expanded incell number over subsequent subculturing steps in vitro, preferablyusing SG medium, which is described below. Furthermore, DAZL-deficienttransgenic rats are obtained from a cross into the desired rat genetic(e.g., Fischer 344) background. The male-sterile recipient animals thusproduced are immuno-compatible with the derived spermatogonial lines.

The phrase “spermatogonial stem cells” in this description denotes stemcells isolated from the testis. Spermatogonial stem cells are incapableof fertilizing an egg cell but can give rise to cells that develop intosperm and that produce viable offspring. Isolated spermatogonial stemcells can be cultured for a prolonged time period without losing theirproperties and can efficiently repopulate the testes of suitablerecipient male animals described, for instance, in Oatley J. M. et al.,Methods Enzymol. 419:259 (2006).

Pursuant to the invention, transplanting cells of a spermatogonial line,as described above, into an immuno-compatible, male-sterile recipientallows the cells to develop into fertilization-competent, haploid malegametes. Since there is negligible endogenous sperm competitiongenerated by the testes of the male-sterile recipient's testes, theinventive approach effects maximal germline transmission of donorhaplotypes from the transplanted spermatogonial cells.

Accordingly, the present invention provides the first vector system foremploying rat spermatogonial lines from in vitro culture in generatingmutant rats on a desired genetic background. The inventive approachallows for more cost-effective production of mutant/transgenic/chimericrat lines and rat cell lines of a pure/inbred genetic background,illustrated by Fischer 344. In relation to the latter, by way ofexample, such production can be accomplished using either the Fischerrat spermatogonia themselves or pluripotent Fischer rat stem cell linesderived directly from the spermatogonia.

A pure genetic background, thus achievable with the present invention,is more desirable than a mixed genetic background for testing theeffects of gene mutations and transgenes in animal studies. Moreover,conventional production of rat lines with loss- or gain-of-functionmutations requires tedious, time-consuming, and prohibitively expensivemicromanipulation of embryos.

By contrast, the inventive approach can use spermatogonia from inbredrats to bypass any requirement for in vitro manipulating of early ratembryos of inbred strains. This reduces the time, cost, and effortrequired to produce mutations in inbred or outbred rat lines.

In particular, the invention enables the production of highly complexspermatogonial libraries, for use as vectors for large-scale productionof mutant rat lines in a desired, preferably inbred genetic background.Such mutant libraries of the invention open the way for producing awider variety of mutant rats, with disrupted genes in an inbred geneticbackground, at orders-of-magnitude faster rates and at a lowerper-animal cost than current approaches can accommodate. Additionally,the invention affords a source of pluripotent rat stem cells, which canbe used in place of rat embryonic stem cells and which is accessiblewithout the need for special skills to manipulate the early rat embryo.

Mutant libraries can be generated, in accordance with the invention,through the use of DNA transposons. DNA transposons can be viewed asnatural gene delivery vehicles that integrate into the host genome via a“cut-and-paste” mechanism. These mobile DNA elements encode atransposase flanked by inverted terminal repeats (ITRs) that contain thetransposase binding sites necessary for transposition. Any gene ofinterest flanked by such ITRs can undergo transposition in the presenceof the transposase supplied in trans.

As noted, a “transposon” is a segment of DNA that can move (transpose)within the genome. A transposon may or may not encode the enzymetransposase, necessary to catalyze its relocation and/or duplication inthe genome. Where a transposon does not code for its transposase enzyme,expression of said enzyme in trans may be required when carrying out themethod of the invention in cells not expressing the relevant transposaseitself Furthermore, a transposon must contain sequences that arerequired for its mobilization, namely the terminal inverted repeatscontaining the binding sites for the transposase. The transposon may bederived from a bacterial or a eukaryotic transposon. Further, thetransposon may be derived from a class I or class II transposon. ClassII or DNA-mediated transposable elements are preferred for gene transferapplications, because transposition of these elements does not involve areverse transcription step, which pertains in transposition of Class Ior retro-elements and which can introduce undesired mutations intotransgenes. For example, see Miller, A. D., RETROVIRUSES 843 (ColdSpring Harbor Laboratory Press, 1997), and Verma, I. M. et al., Nature389:239 (1997).

Transposons also can be harnessed as vehicles for introducing “tagged”genetic mutations into genomes, which makes such genomic sites oftransposon integration/mutation easy to clone and defined at the DNAsequence level. This fact makes transposon-based technology especiallyattractive in cultures of germline stem cells derived from a variety ofmodel species, including the laboratory rat. For example, the firstmutagenesis screens in mammals have established that Sleeping Beauty cangenerate a high number of random mutations in both mouse and ratgerminal cells in vivo. Alternatively, where mutagenic events can firstbe selected and then used to produce experimental animal models, randommutagenesis would be more feasible in tissue culture.

Similarly, transposons can be harnessed as vehicles for introducingmutations into genomes. Specifically, genes may be inactivated bytransposon insertion. For example, such genes are then “tagged” by thetransposable element, which can be used for subsequent cloning of themutated allele. In addition to gene inactivation, a transposon may alsointroduce a transgene of interest into the genome if contained betweenits ITRs. Moreover, to insert or knockin a DNA construct or gene ofinterest into an existing site of transposition, stem cell lines oranimals produced with transposons are designed to contain recognitionsequences (e.g., pLox sties) within the transposon that act assubstrates for DNA recombinase enzymes (e.g., Cre-recombinase). Thiswould allow a gene of interest flanked by compatible recombinaserecognition sequences to be delivered into the cells or animals in transwith a recombinase to catalyze integration of the gene of interest intothe genomic locus of the transposon. The transposon may carry as wellthe regulatory elements necessary for the expression of the transgene,allowing for successful expression of the gene.

Examples of transposon systems that can transpose in vertebrates haverecently became available, such as Sleeping Beauty, piggyBac, Tol2 orFrog Prince. Each transposon system can be combined with any gene trapmechanism (for example: enhancer, promoter, polyA, or slice acceptorgene traps) to generate the mutated gene, as discussed below. SleepingBeauty (SB) and Frog Prince (FP) are Tc1 transposons, whereas piggyBac(PB) was the founder of the PB transposon family and Tol2 is a hATtransposon family member. Both the Sleeping Beauty and the Frog Princetransposon are found in vertebrates as inactive copies, from whichactive transposon systems have been engineered. The Tol2 transposon alsohas been found in vertebrates as an active transposon. The piggyBactransposon was originally found as an active transposon in insects butwas subsequently shown to have high levels of activity in vertebrates,too, as shown in Ding S et al., Cell 122:473 (2005). Each of theseelements has their own advantages; for example, Sleeping Beauty isparticularly useful in region-specific mutagenesis, whereas Tol2 has thehighest tendency to integrate into expressed genes. Hyperactive systemsare available for Sleeping Beauty and piggyBac. Most importantly, thesetransposons have distinct target site preferences, and can thereforemutagenise overlapping, but distinct sets of genes. Therefore, toachieve the best possible coverage of genes, the use of more than oneelement is particularly preferred.

In addition to naturally occurring transposons, modified transposonsystems such as those disclosed in European patent documents EP1594973,EP1594971, and EP1594972 also may be employed in this invention.Preferably, the transposons used for the method of the invention possesshighly elevated transpositional activity.

In a preferred embodiment of the present invention, the transposon is aeukaryotic transposon, such as the Sleeping Beauty transposon, the FrogPrince transposon, the piggyBac transposon, or the Tol2 transposon, asdiscussed above.

The use of gene-trap constructs for insertional mutagenesis in tissueculture, where trapped events can easily be selected for, isadvantageous over the random mutagenesis in animals. Gene trap vectorsreport both the insertion of the transposon into an expressed gene, andhave a mutagenic effect by truncating the transcript through imposedsplicing. Cells selected for a particular gene trap event can be usedfor the generation of animal models lacking this specific geneticfunction.

When transposons are used in insertional mutagenesis screens, transposonvectors typically constitute four major classes of constructs, suitablefor identifying mutated genes rapidly. These contain a reporter gene,which should be expressed depending on the genetic context of theintegration. Specific gene traps include, but are not limited to: (1)enhancer traps, (2) promoter traps, (3) polyA traps, and (4) spliceacceptor traps. In enhancer traps, the expression of the reporterrequires the presence of a genomic cis-regulator to act on an attenuatedpromoter within the integrated construct. Promoter traps contain nopromoter at all. These vectors are only expressed if they land in-framein an exon or close downstream to a promoter of an expressed gene. InpolyA traps, the marker gene lacks a polyA signal, but contains a splicedonor (SD) site. Thus, when integrating into an intron, a fusiontranscript can be synthesized comprising the marker and the downstreamexons of the trapped gene. Slice acceptor gene traps (or exon traps)also lack promoters, but are equipped with a splice acceptor (SA)preceding the marker gene. Reporter activation occurs if the vector isintegrated into an expressed gene, and splicing between the reporter andan upstream exon takes place. The splice acceptor gene trap and polyAgene trap cassettes can be combined. In that case, the marker of thepolyA trap part is amended with a promoter so that the vector also cantrap downstream exons, and both upstream and downstream fusiontranscripts of the trapped gene can be obtained. The foregoingconstructs also offer the possibility to visualize spatial and temporalexpression patterns of the mutated genes by using, e.g., LacZ orfluorescent proteins as a marker gene.

Accordingly, the present invention comprehends a method based on thecombination of transposon-mediated insertional mutagenesis with a tissueculture system, namely, with rat spermatogonial stem cells, which allowsfor the ready generation of in vitro spermatogonial stem cell librariescarrying a large number of different insertion events. Compared toclassical nuclear transfer technologies and in vivo mutagenesis,moreover, this method is less costly and less labor-intensive, and itallows for the selection of the appropriate insertion(s) beforeestablishing the corresponding animal models. Additionally, using thesecells or libraries allows for establishment of a broader variety ofanimal models.

As noted above, the phrase “quantitative trait loci” (QTLs) denotes thelocalization of multiple loci involved in complex diseases andquantitative phenotypes. Certain QTLs demonstrate genetic linkage tomany disease traits that are shared between rats and humans.

A quantitative trait locus may be further mutated using the “localhopping” property of transposons. A transposon insertion site can beremobilized by the expression of the transposase recognizing thetransposon elements responsible for mediating the excision either inspermatogonial stem cells in culture or at a later stage in vivo.

Libraries of spermatogonial cell lines can be generated by isolating andthen pooling individual clonal lines with mutated genes. First,spermatogonial lines are genetically modified with a DNA construct thatharbors a selectable marker, such as a gene encoding resistance to G418.Then, due to stable integration of the DNA construct into differentlocations within the genome, a mixed population of genetically distinctclonal spermatogonial lines is selected using the selectable marker. Bypooling these selected individual clonal lines with mutated genes, alibrary of mutant rat spermatogonia is generated.

In order to transmit stem cell genomes through the rat germline,spermatogonial lines containing gene trap Sleeping Beauty insertions,for example, can be selected in culture and transplanted to repopulatetestes of sterile recipient rats. Thus, the testes of DAZL-deficient andwild-type recipient rats can be transplanted with mixed populations of aselectably-resistant spermatogonial line, such as G418, selected as alibrary estimated to contain ˜200,000 individual clonal lines withtrapped genes (see Example 5 below).

The phrase “selectable marker” is employed here to denote a protein thatenables the separation of cells expressing the marker from those thatlack or do not express it. The selectable marker may be a fluorescentmarker, for instance.

Expression of the marker by cells having successfully integrated thetransposon allows the isolation of these cells using methods such as,for example, FACS (fluorescent activated cell sorting). Alternatively,expression of a selectable marker may confer an advantageous property tothe cell that allows survival of only those cells carrying the gene.

For example, the marker protein may allow for the selection of the cellby conferring an antibiotic resistance to the cell. Consequently, whencells are cultured in medium containing said antibiotic, only cellclones expressing the marker protein that mediates antibiotic resistanceare capable of propagating. By way of illustration, a suitable markerprotein may confer resistance to antibiotics such as ampicillin,kanamycin, chloramphenicol, tetracycline, hygromycin, neomycin ormethotrexate. Further examples of antibiotics are penicillins:ampicillin HCl, ampicillin Na, amoxycillin Na, carbenicillin disodium,penicillin G, cephalosporins, cefotaxim Na, cefalexin HCl, vancomycin,cycloserine. Other examples include bacteriostatic inhibitors such as:chloramphenicol, erythromycin, lincomycin, spectinomycin sulfate,clindamycin HCl, chlortetracycline HCl. Additional examples are markerproteins that allow selection with bactericidal inhibitors such as thoseaffecting protein synthesis irreversibly causing cell death, for exampleaminoglycosides such as gentamycin, hygromycin B, kanamycin, neomycin,streptomycin, G418, tobramycin. Aminoglycosides can be inactivated byenzymes such as NPT II which phosphorylates 3′-OH present on kanamycin,thus inactivating this antibiotic. Some aminoglycoside modifying enzymesacetylate the compounds and block their entry in to the cell. Markerproteins that allow selection with nucleic acid metabolism inhibitorslike rifampicin, mitomycin C, nalidixic acid, doxorubicin HCl,5-fluorouracil, 6-mercaptopurine, antimetabolites, miconazole,trimethoprim, methotrexate, metronidazole, sulfametoxazole are alsoexamples for selectable markers.

The term “rat” refers to a member of the genus Rattus, such as the blackrat, Rattus rattus, and the brown rat, Rattus norvegicus. The laboratoryrat is one of the most extensively studied model organisms for humandisease and, hence, is the major animal model in the initial stages ofdrug development. In contrast to mice, a limitation of the rat model hasbeen the lack of technology for generating “defined” genetic mutants.Such defined genetic mutants, where precise changes to a gene sequenceor function are made without perturbing the rest of the genome, arecritical for determining gene function with a high degree of certaintyand for creating reliable genetic models for human disease. Due to thelack of technology for generating defined mutants in rats, geneticmanipulation approaches were not successful in the rat genome so far andgenome research in the rat to connect genetic backgrounds with certainphenotypes is lagging behind.

Once generated, a spermatogonial cell line is transplanted into amale-sterile recipient rat. In a preferred embodiment of the invention,spermatogonial cell lines are transplanted into transgenically createdmale-sterile recipients. Alternatively, spermatogonial cell lines aretransplanted into recipients that have naturally occurring geneticmutations that cause male-sterility.

Examples of naturally occurring mutations that generate male sterilerats include but are not limited to: rats that have mutations in FKBP6;rats that have altered function of the pituitary-gonadal axis, seeKamtchouing et al. Biol. Reprod. 45:11-19 (1991), for example; and ratsthat have a mutant BIL/1. Illustrative of transgenically generated malesterile rat are the DAZL-deficient transgenic rat and rats that expressthe HSV type 1 thymidine kinase protein.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from thespecified value, as such variations are appropriate to perform thedisclosed methods and maintain the viability of the spermatogonial stemcell line.

As used herein, the terms “fragment” and “fragment of a transposon” aremeant to refer to DNA sequences which are not complete transposon DNAsequences (i.e. full-length DNA sequences) but DNA sequences shorter inlength than the full-length sequence which consist of nucleotidesequences identical to nucleotide sequences of portions of a full-lengthDNA sequence of a transposon. A fragment of a transposon may functionlike a full length DNA. In some embodiments, a fragment of a transposonis a truncated form of the wild-type or full-length DNA transposonsequence. In some embodiments a fragment of a transposon is an internaltandem repeat of the transposon. For example, in some embodiments wherecompositions or methods comprise transplanted haplotypes, the haplotypescomprise fragments of full-length transposons that flank transgenes ofmutated genes of interest. In some embodiments, the transplantedhaplotypes comprise at least one or more of any combination of thefragments of a transposon comprising the following DNA sequences:

Sleeping Beauty 5′ ITR: CAGTTGAAGTCGGAAGTTTACATACACTTAAGTTGGAGTCATTAAAACTCGTTTTTCAACTACTCCACAAATTTCTTGTTAACAAACAATAGTTTTGGCAAGTCAGTTAGGACATCTACTTTGTGCATGACACAAGTCATTTTTCCAACAATTGTTTACAGACAGATTATTTCACTTATAATTCACTGTATCACAATTCCAGTGGGTCAGAAGTTTACATACACTAAGT Sleeping Beauty 3′ ITR:ATTGAGTGTATGTAAACTTCTGACCCACTGGGAATGTGATGAAAGAAATAAAAGCTGAAATGAATCATTCTCTCTACTATTATTCTGATATTTCACATTCTTAAAATAAAGTGGTGATCCTAACTGACCTAAGACAGGGAATTTTTACTAGGATTAAATGTCAGGAATTGTGAAAAAGTGAGTTTAAATGTATTTGGCTAAGGTGTATGTAAACTTCCGACTTCAACTG PiggyBac 5′ ITR:CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATG CATTCTTGAAATATTGCTCTCTCTTTCTAAATAGCGCGAATCCGTCGCTGTGCATTTAGGACATCTCAGTCGCCGCTTGGAGCTCCCGTGAGGCGTGCTTGTCAATGCGGTAAGTGTCACTGATTTTGAACTATAACGACCGCGTGAGTCAAAATGACGCATGATTATCTTTTACGTGACTTTTAAGATTTAACTCATACGATAATTATATTGTTATTTCATGTTCTACTTACGTGATAACTTATTATATATA TATTTTCTTGTTATAGATATC(minimal sequence is underlined and bold, i.e., first 35 bp) PiggyBac 3′ITR: TAAAAGTTTTGTTACTTTATAGAAGAAATTTTGAGTTTTTGTTTTTTTTTAATAAATAAATAAACATAAATAAATTGTTTGTTGAATTTATTATTAGTATGTAAGTGTAAATATAATAAAACTTAATATCTATTCAAATTAATAAATAAACCTCGATATACAGACCGATAAAACA CATGCGTCAATTTTACGCATGATTATCTTTAACGTACGTCACAATATGATTATCTTTCTAGGG(minimal sequence is underlined and bold, i.e., first 35 bp)

In some embodiments, the only transposon fragment in the transplantedhaplotype consists of a PiggyBac 5′ ITR and a PiggyBac 3′ ITR. In someembodiments, the only transposon fragment in the transplanted haplotypeconsists of a Sleeping Beauty 5′ ITR and a Sleeping Beauty 3′ ITR. Insome embodiments, the transplanted haplotype comprises a transgeneflanked by a PiggyBac 5′ ITR and a PiggyBac 3′ ITR. In some embodiments,the transplanted haplotype comprises a transgene flanked by a SleepingBeauty 5′ ITR and a Sleeping Beauty 3′ ITR. In some embodiments, thetransplanted haplotype comprises a transgene flanked by the followingsequences: 5′ CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATG and on the 3′ end(from 5′ to 3′): CATGCGTCAATTTTACGCATGATTATCTTTAACGTACGTCACAATATGATTATCTTTCTAGGG. In some embodiments, the transplanted haplotype comprises ahyperactive transposon.

The present invention also provides a composition that comprises a cellculture medium, glial cell-derived neurotrophic factor (GDNF),Fibroblast Growth Factor-2 (FGF2), and B27-minus vitamin A supplementsolution. In some embodiments, the composition further comprises any oneor more of Ham's F12 nutrient mixture, 2-mercaptoethanol, andL-glutamine. In some embodiments, the cell culture medium is Dulbecco'sModified Eagle Medium (DMEM). In addition, 2-mercaptoethanol andL-glutamine can be substituted by similar functioning compounds wellknown to the skilled artisan.

In some embodiments, the composition described above can comprise fromabout 10 ng/ml to about 30 ng/ml GDNF, from about 10 ng/ml to about 30ng/ml FGF2, and about a 1× concentration of B27-minus vitamin Asupplement solution. In some embodiments, the composition can comprise a1:1 ratio of cell culture medium, such as DMEM, to Ham's F12 nutrientmixture. The composition can also comprise from about 50 to about 120 μM2-mercaptoethanol, and from about 3 to about 10 mM L-glutamine.

In some embodiments, the composition described above can comprise a 1:1ratio of DMEM to Ham's F12 nutrient mixture, 20 ng/ml GDNF, 25 ng/mlFGF2, 100 μM 2-mercaptoethanol, 6 mM L-glutamine, and a 1× concentrationof B27-minus vitamin A supplement solution. In some embodiments, thecomposition described above can comprise a 1:1 ratio of DMEM to Ham'sF12 nutrient mixture, about 20 ng/ml GDNF, about 25 ng/ml FGF2, about100 μM 2-mercaptoethanol, about 6 mM L-glutamine, and a 1× concentrationof B27-minus vitamin A supplement solution.

The present invention also provides a male rat of a predeterminedgenetic background comprising a transplanted haplotype derived from arat spermatogonial stem cell line, wherein the rat is sterile absent thepresence of the transplanted haplotype. In some embodiments, the malerat is DAZL-deficient. In some embodiments, the male rat expresses asmall hairpin RNA transgene that degrades DAZL mRNA. In someembodiments, the predetermined genetic background is Sprague Dawley orFisher 344. In some embodiments, the transplanted haplotype comprises aninternal tandem repeat (ITR) from a transposon. In some embodiments, thetransplanted haplotype comprises an ITR from a transposon selected fromthe piggyBac ITR, Sleeping Beauty ITR, or a combination thereof.

The present invention also provides a rat spermatogonial stem cell lineof a predetermined genetic background. In some embodiments, thepredetermined genetic background of the rat spermatogonial stem cellline is Sprague-Dawley. In some embodiments, the predetermined geneticbackground of the rat spermatogonial stem cell line is Sprague-Fisher344. In some embodiments, the transplanted haplotype of the ratspermatogonial stem cell line comprises an internal tandem repeat (ITR)from a transposon. The ITR consists of a 5′ sequence and a 3′ sequenceflanking a gene of interest. In some embodiments, the transplantedhaplotype of the rat spermatogonial stem cell line comprises at leastone ITR from a transposon selected from the piggyBac ITR, SleepingBeauty ITR, Tol2 ITR, Frog Prince ITR or a combination thereof. In someembodiments, the rat spermatogonial stem cell line can be cultured fromabout 150 days to about 205 days. In some embodiments, the ratspermatogonial stem cell line has a doubling time of about 8 or 9 days.In some embodiments, the rat spermatogonial stem cell line has adoubling time of no less than 8.4 days. In some embodiments, the ratspermatogonial stem cell line has a doubling time of about 8.4 days. Insome embodiments, the rat spermatogonial stem cell line expands no lessthan about 20,000 times as compared to the number of cells seeded inculture. In some embodiments, the rat spermatogonial stem cell lineexpands no less than about 2,000,000 times as compared to the number ofcells seeded in culture. In some embodiments, the rat spermatogonialstem cell line expands no less than about 30,000 times as compared tothe number of cells seeded in culture. In some embodiments, the ratspermatogonial stem cell line expands no less than about 50,000 times ascompared to the number of cells seeded in culture. In some embodiments,the rat spermatogonial stem cell line expands no less than about 80,000times as compared to the number of cells seeded in culture. In someembodiments, the rat spermatogonial stem cell line expands no less thanabout 100,000 times as compared to the number of cells seeded inculture. In some embodiments, the rat spermatogonial stem cell lineexpands no less than about 200,000 times as compared to the number ofcells seeded in culture. In some embodiments, the rat spermatogonialstem cell line expands no less than about 300,000 times as compared tothe number of cells seeded in culture. In some embodiments, the ratspermatogonial stem cell line expands no less than about 300,000 timesas compared to the number of cells seeded in culture. In someembodiments, the rat spermatogonial stem cell line expands no less thanabout 400,000 times as compared to the number of cells seeded inculture. In some embodiments, the rat spermatogonial stem cell lineexpands no less than about 500,000 times as compared to the number ofcells seeded in culture. In some embodiments, the rat spermatogonialstem cell line expands no less than about 600,000 times as compared tothe number of cells seeded in culture. In some embodiments, the ratspermatogonial stem cell line expands no less than about 700,000 timesas compared to the number of cells seeded in culture. In someembodiments, the rat spermatogonial stem cell line expands no less thanabout 900,000 times as compared to the number of cells seeded inculture. In some embodiments, the rat spermatogonial stem cell lineexpands no less than about 1,000,000 times as compared to the number ofcells seeded in culture.

In some embodiments, the rat spermatogonial stem cell line expands noless than about 1,500,000 times as compared to the number of cellsseeded in culture. In some embodiments, the rat spermatogonial stem cellline expands about 2,0000,000 times as compared to the number of cellsseeded in culture.

In some embodiments, the rat spermatogonial stem cell line can be frozenat about −196 degrees Celsius and possess at least 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 95% cell viability upon thawing and subsequentculturing.

The present invention also comprises composition that comprises aspermatogonial stem cell of a predetermined background and culturemedium. In some embodiments, the culture medium comprises glialcell-derived neurotrophic factor (GDNF), Fibroblast Growth Factor-2(FGF2), and B27-minus vitamin A supplement solution. In someembodiments, the composition further comprises any one or more of Ham'sF12 nutrient mixture, 2-mercaptoethanol, and L-glutamine. In someembodiments, the cell culture medium is Dulbecco's Modified Eagle Medium(DMEM). In addition, 2-mercaptoethanol and L-glutamine can besubstituted by similar functioning compounds well known to the skilledartisan. In some embodiments, the composition can comprise from about 10ng/ml to about 30 ng/ml GDNF, from about 10 ng/ml to about 30 ng/mlFGF2, and about a 1× concentration of B27-minus vitamin A supplementsolution. In some embodiments, the composition can comprise a 1:1 ratioof cell culture medium, such as DMEM, to Ham's F12 nutrient mixture. Thecomposition can also comprise from about 50 to about 120 μM2-mercaptoethanol, and from about 3 to about 10 mM L-glutamine. Thecomposition supports in vitro culturing of spermatogonial stem cells.The spermatogonial stem cell of a predetermined background can compriseany or all of the features described herein for a spermatogonial stemcell. In some embodiments, the composition can comprise about 20 ng/mlGDNF, about 20 ng/ml FGF2, and about a 1× concentration of B27-minusvitamin A supplement solution. In some embodiments, the composition cancomprise a 1:1 ratio of cell culture medium, such as DMEM, to Ham's F12nutrient mixture.

The present invention also provides a method of introducing a mutationor transgene of interest into the genome of a male rat of apredetermined genetic background, wherein said rat is sterile absent thepresence of a transplanted haplotype comprising the steps of: (a)culturing an isolated rat spermatogonial stem cell line of apredetermined genetic background; and (b) transplanting the ratspermatogonial stem cell line of a predetermined genetic background intothe testes of the male rat, wherein the haplotype comprises a transposonsequence or a fragment thereof.

The present invention also provides a method of introducing a transgeneor mutated gene of interest into the genome of a male rat of apredetermined genetic background, wherein said rat is sterile absent thepresence of a transplanted haplotype comprising:

(a) providing a rat spermatogonial stem cell line of a predeterminedgenetic background; (b) genetically modifying the rat stem cell linewith a transposon; and

(c) transplanting the rat spermatogonial stem cell line of apredetermined genetic background into the testes of the male rat.

In some embodiments, the transplanted haplotype comprises an internaltandem repeat (ITR) from a transposon. In some embodiments, thetransplanted haplotype comprises an ITR from a transposon selected fromthe piggyBac ITR, Sleeping Beauty ITR, or a combination thereof. In someembodiments, the transplanted haplotype comprises a fragment of atransposon sequence comprising the following two sequences:CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATG andCATGCGTCAATTTTACGCATGATTATCTTTAACGTACGTCACAATATGATTATC TTTCTAGGG. Insome embodiments, the transplanted haplotype comprises at least twononcontiguous fragments of a transposon sequence comprising thefollowing two sequences:

CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATGCATTCTTGAAATATTGCTCTCTCTTTCTAAATAGCGCGAATCCGTCGCTGTGCATTTAGGACATCTCAGTCGCCGCTTGGAGCTCCCGTGAGGCGTGCTTGTCAATGCGGTAAGTGTCACTGATTTTGAACTATAACGACCGCGTGAGTCAAAATGACGCATGATTATCTTTTACGTGACTTTTAAGATTTAACTCATACGATAATTATATTGTTATTTCATGTTCTACTTACGTGATAACTTATTATATATATATTTTCTTGTT ATAGATATC andTAAAAGTTTTGTTACTTTATAGAAGAAATTTTGAGTTTTTGTTTTTTTTTAATAAATAAATAAACATAAATAAATTGTTTGTTGAATTTATTATTAGTATGTAAGTGTAAATATAATAAAACTTAATATCTATTCAAATTAATAAATAAACCTCGATATACAGACCGATAAAACACATGCGTCAATTTTACGCATGATTATCTTTAACGTACGTCACAATATGATTATCTTTCTAGGG.In some embodiments, wherein the transplanted haplotype comprises afragment of a transposon sequence consisting of the PiggyBac 5′ ITR andthe PiggyBac 3′ ITR. In some embodiments, the transplanted haplotypecomprises a fragment of a transposon sequence consisting of the SleepingBeauty 5′ ITR and the Sleeping Beauty 3′ ITR.

Those having ordinary skill in the art can readily design and producemutations in the DNA sequences above having substantially identicalnucleotide sequences of transposons or transposon ITR with deletionsand/or insertions and/or conservative substitutions of nucleotide basepairs. Such substitutions are well-known and are based the secondarystructure and free energy of the DNA sequence and structuralcharacteristics of each nucleotide base. Derivatives include fragmentsof transposons with deletions and/or insertions and/or conservativesubstitutions. In some embodiments, the transplanted haplotype comprisesa fragment of a transposon sequence consisting of a DNA sequence that is90% homologous to the PiggyBac 5′ ITR and a DNA sequence that is 90%homologous the PiggyBac 3′ ITR. In some embodiments, the transplantedhaplotype comprises a fragment of a transposon sequence consisting of aDNA sequence that is 95% homologous to the PiggyBac 5′ ITR and a DNAsequence that is 95% homologous the PiggyBac 3′ ITR. In someembodiments, the transplanted haplotype comprises a fragment of atransposon sequence consisting of a DNA sequence that is 98% homologousto the PiggyBac 5′ ITR and a DNA sequence that is 98% homologous thePiggyBac 3′ ITR. In some embodiments, the transplanted haplotypecomprises a fragment of a transposon sequence consisting of a DNAsequence that is 99% homologous to the Sleeping Beauty 5′ ITR and a DNAsequence that is 99% homologous the Sleeping Beauty 3′ ITR. In someembodiments, the transplanted haplotype comprises a fragment of atransposon sequence consisting of a DNA sequence that is 90% homologousto the Sleeping Beauty 5′ ITR and a DNA sequence that is 90% homologousthe Sleeping Beauty 3′ ITR. In some embodiments, the transplantedhaplotype comprises a fragment of a transposon sequence consisting of aDNA sequence that is 95% homologous to the Sleeping Beauty 5′ ITR and aDNA sequence that is 95% homologous the Sleeping Beauty 3′ ITR. In someembodiments, the transplanted haplotype comprises a fragment of atransposon sequence consisting of a DNA sequence that is 98% homologousto the Sleeping Beauty 5′ ITR and a DNA sequence that is 98% homologousthe Sleeping Beauty 3′ ITR. In some embodiments, the transplantedhaplotype comprises a fragment of a transposon sequence consisting of aDNA sequence that is 99% homologous to the Sleeping Beauty 5′ ITR and aDNA sequence that is 99% homologous the Sleeping Beauty 3′ ITR.

In some embodiments, the transplanted haplotype comprises a fragment ofa transposon sequence comprising a DNA sequence that is 90% homologousto the PiggyBac 5′ ITR and a DNA sequence that is 90% homologous thePiggyBac 3′ ITR. In some embodiments, the transplanted haplotypecomprises a fragment of a transposon sequence comprising a DNA sequencethat is 95% homologous to the PiggyBac 5′ ITR and a DNA sequence that is95% homologous the PiggyBac 3′ ITR. In some embodiments, thetransplanted haplotype comprises a fragment of a transposon sequencecomprising a DNA sequence that is 98% homologous to the PiggyBac 5′ ITRand a DNA sequence that is 98% homologous the PiggyBac 3′ ITR. In someembodiments, the transplanted haplotype comprises a fragment of atransposon sequence comprising a DNA sequence that is 99% homologous tothe Sleeping Beauty 5′ ITR and a DNA sequence that is 99% homologous theSleeping Beauty 3′ ITR. In some embodiments, the transplanted haplotypecomprises a fragment of a transposon sequence comprising a DNA sequencethat is 90% homologous to the Sleeping Beauty 5′ ITR and a DNA sequencethat is 90% homologous the Sleeping Beauty 3′ ITR. In some embodiments,the transplanted haplotype comprises a fragment of a transposon sequencecomprising a DNA sequence that is 95% homologous to the Sleeping Beauty5′ ITR and a DNA sequence that is 95% homologous the Sleeping Beauty 3′ITR. In some embodiments, the transplanted haplotype comprises afragment of a transposon sequence comprising a DNA sequence that is 98%homologous to the Sleeping Beauty 5′ ITR and a DNA sequence that is 98%homologous the Sleeping Beauty 3′ ITR. In some embodiments, thetransplanted haplotype comprises a fragment of a transposon sequencecomprising a DNA sequence that is 99% homologous to the Sleeping Beauty5′ ITR and a DNA sequence that is 99% homologous the Sleeping Beauty 3′ITR.

In some embodiments, the male rat of the composition and method claimsis transplanted via injection of the cells. In some embodiments, themale rat is DAZL-deficient. In some embodiments, the male rat expressesa small hairpin RNA transgene that degrades DAZL mRNA. In someembodiments, the predetermined genetic background is Sprague Dawley orFisher 344. In some embodiments, the predetermined genetic background isan outbred strain of rat. In some embodiments, the predetermined geneticbackground is an inbred strain of rat. In some embodiments, thetransplanted stem cell line comprises an internal tandem repeat (ITR)from a transposon. In some embodiments, the transplanted stem cell linecomprises an ITR from a transposon selected from the piggyBac ITR,Sleeping Beauty ITR, or a combination thereof. In some embodiments, themale rat of a predetermined genetic background comprises a transgene ormutation. In some embodiments, the male rat of a predetermined geneticbackground comprises a transposon. In some embodiments, the male rat ofa predetermined genetic background comprises a transposon ITR. In someembodiments, the rat spermatogonial stem cell line comprises at leastone ITR from a transposon selected from the piggyBac ITR, SleepingBeauty ITR, Tol2 ITR, Frog Prince ITR or a combination thereof. In someembodiments, the male rat of a predetermined genetic background at leastone ITR from a transposon selected from the piggyBac ITR, SleepingBeauty ITR, Tol2 ITR, Frog Prince ITR or a combination thereof.

The present invention is further described by reference to the followingexamples, which are illustrative only and not limiting of the invention.

Example 1 Generation of DAZL-Deficient Rats on a Fischer F344 GeneticBackground

Male DAZL-deficient rats are sterile, whereas female DAZL-deficient ratsremain fully fertile. Somatic testis cells in DAZL-deficient rats areable to support full development of spermatozoa from healthy donorspermatogonial stem cell lines derived from normal Sprague Dawley rats,thus the effects of DAZL on disrupting sperm cell development arerestricted to the germ-line. Importantly, the absence of endogenoussperm production by DAZL-deficient rats allows the sperm produced fromthe transplanted, donor spermatogonial stem cell lines to develop andfunction within an environment devoid of any competition from the hostsgermline. The selected, genetically modified haploid gametes can then beused to produce mutant rats through natural mating of recipients, or byassisted reproduction techniques.

To use the DAZL-deficient recipient rat model as a strategy to enrichfor fully functional Fischer F344 Rat spermatogonial stem cell linesthat can develop into fertilization competent gametes, DAZL-deficientrats of a predominantly Fischer F344 genetic background were firstgenerated to avoid immuno-compatibility barriers that exist between theSprague Dawley and Fischer F344 rat lines. Because initial existingstrains of DAZL-deficient rats were on a Sprague Dawley rat geneticbackground, female DAZL-deficient rats were first bred with pure maleFischer F344 rats (Harlan, Inc). Based on Mendelian inheritance ofmaternal and paternal genes, the progeny (F1 progeny) produced from thiscross yielded rats that consisted of ˜half Fischer F344 and ˜halfSprague Dawley genetic background. Accordingly, ˜50% of Female F1progeny from this first out-cross are also DAZL-deficient due toinheritance of the DAZL-shRNA transgene. DAZL-deficient F1 female ratswere then back-crossed with pure, male Fischer F344 rats (Harlan, Inc)again to enrich for the Fischer F344 genetic background by ˜75% in theF2 progeny. At this point, F2 DAZL-deficient males and females should beenriched for Fischer F344 immuno-compatibility factors by at least50-75%. DAZL-deficient F2 males of this hybrid background can now serveas recipients for pure donor Fischer F344 rat spermatogonial stem celllines. Also, the F2 females generated can be used to further enrich forthe Fischer F344 immuno-compatibility factors in DAZL-deficient rats byadditional back-crossing with pure, male Fischer F344 rats (Harlan, Inc)until near genetic homogeneity with the derived Fischer F344 ratspermatogonial lines. The number of back-crosses to achieve optimalspermatozoon development from donor Fischer F344 spermatogonial stemcell lines is not currently known, but could be maximal after theinitial cross.

Example 2 Nutrient Mixture for Culturing Rat Spermatogonial Stem Cells

SG medium is an effective and efficient nutrient mixture for growing ratspermatogonial stem cells. SG medium is composed of Dulbecco modifiedEagle medium:Ham F12 nutrient mixture (1:1), 20 ng/ml GDNF, 25 ng/mlFGF2, 100 μM 2-mercaptoethanol, 6 mM L-glutamine and a 1× concentrationof the B27-Minus Vitamin-A Supplement solution. With the use of SGmedium, six spermatogonial lines were derived from the testes of six,separate Sprague Dawley rats. After proliferating over a 120 day periodin SG medium, stem cells within the spermatogonial cultures effectivelyregenerated spermatogenesis in testes of busulfan-treated recipientrats, which transmitted the donor cell haplotype to greater than 75% ofprogeny by natural breeding. Sub-culturing in SG medium did not requireprotease treatment, and was achieved by passaging the loosely boundspermatogonial cultures at 1:3 dilutions onto fresh mono-layers ofirradiated, DR4 mouse fibroblasts every 12 days. Spermatogonial linesderived and propagated using SG medium were characterized as homogenouspopulations of ZBTB 16⁺, DAZL⁺ cells endowed with spermatogonial stemcell potential.

Materials

Dispase, rat-tail collagen I-coated culture dishes and gelatin-coatedculture dishes were from, Fisher, Inc. Phosphate buffered saline (PBS),nonessential amino acids, Minimum Essential Medium (MEM) vitaminsolution, 1-glutamine solution, trypsin-EDTA solutions (0.05% wt/voltrypsin with 0.2 gaiter EDTA.4Na; or 0.25% wt/vol trypsin with 0.38g/liter EDTA.4Na), antibiotic-antimycotic solution (catalogue15240-062), Hoechst 33342 and AlexaFlour-594-conjugated,goat-anti-rabbit and goat-anti-mouse IgGs were from Invitrogen. Bovineserum albumin (BSA) and dimethyl-sulfoxide (DMSO) were from Calbiochem.Fetal bovine serum (FBS) for mouse embryonic fibroblast (MEF) medium wasfrom Hyclone (catalogue SH30071.03). Blocking reagent was from RocheApplied Biosciences. Mouse laminin, sodium bicarbonate, trypan blue,Dulbecco's Modified Eagle's Medium (DMEM, Cat#D5648), and DMEM:Ham's F12(1:1) Nutrient Mixture (DMEM:Ham's F12, Cat#D8437) were from Sigma. SeeSigma Cat#D5648 and D8437 Product Information Sheet.

Animal Care and Use

Protocols for the use of rats in this study were approved by theInstitutional Animal Care and Use Committee (IACUC) at UT-SouthwesternMedical Center in Dallas. Rats used for this study were housed inindividually ventilated, Lab Products 2100 cages in a dedicated roomwith atmosphere controls set to 72° F., 45-50% humidity during a 12 hourlight/dark (i.e. Light cycle=6:00 am-6:00 pm, Central Standard Timeadjusted for daylight savings time). Rats were fed Harlan TekladIrradiated 7912, LM-485 Mouse/Rat Diet, 5% fat Diet and a continuoussupply of reverse osmosis water.

Isolating Enriched Fractions of Undifferentiated Spermatogonia

Seminiferous tubules were isolated from testes of 23-24 day old WTSprague Dawley (SD) rats (Hsd:Sprague Dawley SD, Harlan, Inc.) orhomozygous SD-Tg(Gt(ROSA)26Sor-EGFP)2-4Reh transgenic rats. Rats of theSD-Tg(Gt(ROSA)26Sor-EGFP)2-4Reh line were produced by pronuclearinjection and are referred to as GCS-EGFP rats because they exhibit germcell specific (GCS) expression of enhanced green fluorescent protein(EGFP). The tubules were enzymatically and mechanically dissociated intoa cellular suspension to generate cultures of testis cells inserum-containing medium, as described, except that a medium volume of 10ml/rat was applied for all centrifugation and filtration steps. Thetestis cell cultures were then used to isolate enriched populations oflaminin-binding spermatogonia following previously established methods,which describe how to first remove >95% of somatic testis cells from thegerm cell population by negative selection on plastic and collagen,before positive selection for the spermatogonial stem cells based ontheir ability to bind to laminin. By this procedure, the freshlyisolated laminin-binding germ cell population contains >90%undifferentiated, type A spermatogonia (ZBTB16⁺, DAZL⁺) in the single(˜88%) or paired (˜12%) cell state. Also, it should be noted thatfractions of laminin-binding spermatogonia isolated by this procedurecontain ˜4% somatic cells, and ˜5% differentiating spermatogonia plusspermatocytes. In this study, a single rat of this age range yielded3.62×10⁵±0.93×10⁵ (SD, n=6 rats) laminin-binding spermatogonia, ascompared to yields reported for this procedure when scaled forprocessing pools of testes from multiple rats at 22-24 days of age(i.e., 1.98×10⁵±0.56×10⁵ cells/rat, SD, n=34 primary cultures).

Derivation of Spermatogonial Lines

To derive rat spermatogonial stem cell lines, freshly isolatedlaminin-binding spermatogonia from individual rats were platedseparately into gelatin-coated wells (3.5 cm) of a culture plate at˜1.9×10⁴ cells/cm² in 0.37 ml/cm² of Spermatogonial Medium (SG Medium).Components of SG Medium are presented in Table 2 and Table 3. Asreported for this procedure using SA Medium, which lacks both serum andvitamin-A, spermatogonia cultured on gelatin using SG Medium wereobserved loosely bound to the culture plate, bound to residual adherentsomatic testis cells, and in suspension; many of the spermatogonia insuspension adhered to each other as cellular “clusters” of variablesize. In contrast, the small fraction of contaminating somatic testiscells attached avidly and spread out on the gelatin matrix. After aninitial selection for 40-48 hours on the gelatin-coated plates,spermatogonia in suspension (i.e., including loosely boundspermatogonia), were harvested free from the contaminating somatictestis cells by pipetting. Harvested spermatogonia were pelleted at200×g for 4 minutes, the supernatant was discarded and the cellularpellet was suspended in SG Medium and plated into fresh gelatin-coatedwells (3.5 cm) for an additional 72-96 hours. After this point (i.e.,after depletion of essentially all somatic testis cells), suspensions ofspermatogonia from each rat that survived through the final selectionsteps on gelatin were harvested into fresh SG Medium and passaged into2.2 cm culture wells (i.e., 12-well culture dish) containing feederlayers of irradiated mouse embryonic fibroblasts (MEFs) Methods forpreparing MEF feeder layers are described below. The initial passage ofspermatogonial cultures after their plating onto MEF feeder layersrequired a 1:1 to 1:2 split into the same size wells at 14-21 days aftertheir initial seeding onto the MEFs. In this situation, becauseirradiated MEF feeder layers are not as effective after 14 days inculture, fresh MEFs (2×10⁴/crn²) were “spiked” into the on-goingspermatogonial cultures on day 12-14 so as to by-pass the need topassage the spermatogonia before expanding to larger numbers. However,once established by the second or third passage on MEFs, cultures ofspermatogonia were passaged at ˜1:3 dilutions onto a fresh monolayer ofMEFs every 10-14 days at ˜3×10⁴ cells/cm² for over 5 months (˜12passages). For passaging, cultures were first harvested by gentlypipetting them free from the MEFs. After harvesting, the “clusters” ofspermatogonia were dissociated by gentle trituration with 20-30 strokesthrough a p1000 pipet in their SG culture medium. The dissociated cellswere pelleted at 200×g for 4 minutes and the number of cells recoveredduring each passage was determined by counting them on a Hemocytometer(Note: spermatogonial clusters were not disrupted for counting until thesecond passage on MEFs). As verified by expression of the GCS-EGFPmarker transgene, spermatogonia were easily distinguished duringcounting as the predominant population of smaller, round cells withsmooth surfaces, as compared to occasionally observed, larger and oftenirregular shaped irradiated MEFs. All culture steps for deriving andpropagating spermatogonial lines when in SG Medium were performed at 37°C./5% CO₂. The doubling time for the number of GCS-EGFP⁺ cells thatcould be harvested from cultures of each spermatogonial line aftersubsequent passages between days 30 and 150 in culture on MEFs werecalculated by non-linier regression analysis using the least squares fitmodel set for automatic outlier exclusion provided as the ExponentialGrowth Equation in the GraphPad Prism program (Version 5.01, GraphPadSoftware, Inc.).

Preparation of Fibroblast Feeder Layers

Primary stocks of DR4MEFs were purchased from ATCC, Inc., and expandedin DMEM supplemented 1.5 g/l sodium bicarbonate, 15% heat-inactivatedFBS (MEF medium) at 37° C./5% CO₂ for up to 4 passages following theirthawing and initial plating (i.e. passage 0) from the vial received fromthe manufacturer. Following expansion to passages 3 and 4, secondarystocks of MEFs were irradiated (120 Gy) and then cryo-preserved inliquid nitrogen for future use by the manufacturer's protocol. Prior touse for culture with spermatogonia, the MEFs were thawed and plated intogelatin-coated dishes (4.5×10⁴ cells/cm²) in MEF medium for 16-48 hr,rinsed 1× with PBS and then pre-incubated in SG Medium for an additional16-48 hr. The SG Medium used for pre-incubation was then discarded andspermatogonia were passaged onto the MEFs in fresh SG Medium.

Germ Cell Transplantation and Progeny Genotyping

WT Sprague Dawley rats at 12 days of age were injected (i.p.) with 12.5mg/kg busulfan (4 mg/ml in 50% DMSO) and then used as recipient males at24 days of age. Busulfan is a spermatogonial toxin commonly used to killspermatogonia in recipient rat testes prior to transplantation becauseit increases the colonization efficiency by the donor stem cells. Donorcells were loaded into injection needles fashioned from 100 μl glasscapillary tubes at a concentration of 3×10⁵ cells/65 μl SG Mediumcontaining 0.04% (wt/vol) trypan blue and then the entire volume wastransplanted into the seminiferous tubules of anesthetized rats byretrograde injection through the rete testes. Recipient malestransplanted with GCS-EGFP spermatogonia were paired with wild-typefemale Sprague Dawley of similar age at 75 days post transplantation.Transgenic rat progeny from wild-type recipients and wild-type femaleswere determined by qtPCR analysis of genomic DNA using primers specificto the GCS-EGFP transgene and the 18S ribosomal subunit; relativetransgene copy number in F2 progeny from hemizygous crosses wereverified by Southern dot blot hybridization analysis of the genomic DNAusing a probe specific for the GCS-EGFP transgene. Genotyping resultswere also confirmed in representative progeny by direct visualization oftransgene expression in testes and ovaries using a Nikon SMZ 1500fluorescence stereomicroscope.

Immunocytochemistry

Cultures of germ cells (2 cm²) were washed twice with DMEM:Ham's F12medium (0.6 ml/well) and then fixed for 7.5 mM with 4% paraformaldehyde,0.1M sodium phosphate, pH 7.2 (0.4 ml/well). After fixation the cellswere washed 3 times with PBS (0.6 ml/well) and then incubated for 15 minin PBS containing 0.1% (v/v) triton-X 100 (0.4 ml/well). The cells werethen washed 3 times in PBS (0.6 ml/well) and non-specific,protein-binding sites were blocked by incubating the cells in 0.1% w/vblocking reagent (0.4 ml/well, Roche, Inc.) for 1.5 hr at 22-24° C. Theblocking reagent was then removed and the cells were incubated for 16 hrat 22-24° C. in primary antibodies (0.4 ml/well). The mouse, anti-humanZBTB 16 IgG (Calbiochem, Inc.) and the purified non-immune mouse IGHG1(Santa Cruz, Inc) fractions were each diluted to 1 μl/ml in blockingreagent. The anti-DAZL-3 IgG and the preimmune-3 IgG fractions werediluted to 250 ng/ml in blocking reagent. Following incubation inprimary antibodies, the cells were washed 3 times for 5 min with 0.6ml/well 10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH 7.5 (TBST) toremove unbound IgG. The cells were then incubated for 40 min at 22-24°C. in conjugated, secondary antibody (0.4 ml/well) diluted to 4 μg/ml inPBS containing 5 μg/ml Hoechst 33342 dye. Following incubation insecondary antibodies, the cells were washed 3 times for 5 min with TBST(0.6 ml/well) to remove unbound IgG and dye prior to viewing in freshPBS (0.4 ml/well) using an inverted Olympus IX70 microscope (Olympus,Inc.).

Formula and Effects of a New Spermatogonial Culture Medium:

A DMEM:Hams-F12 nutrient mixture supplemented with GDNF, bFGF,B-27-Minus Vitamin-A, L-glutamine and 2-mercaptoethanol was found tosupport the continued propagation (>2 million-fold expansion in cellnumber) of a previously established rat spermatogonial line on MEFsfollowing its initial derivation, propagation and cryo-preservation inSA medium. The newly formulated spermatogonial medium was termed SGmedium, and further eliminated the need to add MEM-vitamins, estradiol,pyruvate, lactate, ascorbate, non-essential amino acids, glucose andStemPro supplement. The SG medium was also used at a 100% success rateto derive new proliferating spermatogonial lines from individualwildtype (n=3) and homozygous GCS-EGFP transgenic rats (n=3) on aSprague Dawley background by our previously established methods (SeeHamra et al., Proc Natl Acad Sci USA 2005; 102:17430-17435), but withoutthe need to further enrich the starting spermatogonial population byflow cytometry or magnetic cell sorting techniques. The proliferatinggermlines derived in SG medium were characterized as undifferentiatedspermatogonia based on co-expression of the marker proteins, PLZF andDAZL, and their ability to effectively colonize the seminiferous tubulesof busulfan-treated rats. The lines were sub-cultured in SG medium bypipetting, and did not require protease treatment. The newly derivedlines displayed doubling times of 8.4±0.2 days (mean±SD, n=4) whenexponential growth curves were fit between culture days 30 and 150 aftertheir initial seeding onto MEFs in SG medium as freshly isolatedlaminin-binding spermatogonia. For comparison, spermatogonial linesderived and propagated in SA medium displayed doubling times of 6.5±1.8days (mean±SD, n=4) when analyzed between culture days 30 and 150 aftertheir initial seeding onto MEFs.

To determine the spermatogenic potential of the new rat spermatogoniallines (RSGL) that were derived using SG medium, RSGL-GCS9 and RSGL-GCS10from GCS-EGFP rats were propagated for 111 and 120 days in culture,respectively, over a total of 9 to 10 passages prior to beingtransplanted into testes of busulfan-treated, wildtype rats at ˜3×10⁵cells/testis (i.e. after expanding in cell number by >20.000-fold aftertheir initial seeding onto MEFs). When the recipients of RSGL-GCS9 andRSGL-GCS10 were paired with wild-type females at 75 dayspost-transplantation, they yielded 78.9±10.4% and 67.2±16.4% germlinetransmission, respectively, from spermatozoa produced by the donor stemcells (GCS9 recipients: mean±SD, n=19 litters, 193 total pups; GCS10recipients: mean±SD, n=9 litters, 107 total pups). Germline transmissionof the donor cell haplotype was based on inheritance of the GCS-EGFPtransgene by F1 progeny. Resulting non-Mendelian ratios (i.e. <100%transgenic F1 progeny) were due to competition from residual wildtypespermatozoa produced by the recipients. However, transmission of theGCS-EGFP transgene to F2 progeny from crosses between hemizygous F1cousins did yield Mendelian ratios (wildtype=21%, hemizygous=51%,homozygous=28%; n=3 litters, 47 total pups).

Testes from recipients transplanted with RSGL-GCS9 and RSGL-GCS10 werenext analyzed histologically for long-term, spermatogenesis colonyforming potential. When evaluated at 206-263 days followingtransplantation, 92.5±2.4% (mean±SEM, n=4) and 81.5±9.5% (mean±SEM, n=3)of seminiferous tubule cross sections that were colonized by RSGL-GCS9and RSGL-GCS10, respectively, showed development of EGFP+ spermatogoniato the elongating spermatid stage. Thus, spermatogonial lines derived inSG medium were classified as essentially pure cultures ofundifferentiated spermatogonia containing fully functional sperm stemcells.

Example 3 Deriving Spermatogonial Lines from Inbred Fischer, F344 NHsdRats

Total laminin-binding (LB) spermatogonia (2.3×10⁶) were isolated fromthe seminiferous tubules of 10 inbred Fischer Rats (F344 NHsd Rats,Harlan, Inc) at 22 days of age. The isolated spermatogonia were thenused to derive lines of proliferating spermatogonia in SpermatogonialCulture Medium (SG Medium) using established methods. In brief,following isolation, LB spermatogonia were plated equally into 6,gelatin-coated wells (9.6 cm² wells) of a 6-well culture plate. Thecells were harvested by gentle pipetting, pelleted at 200×g for 4 min,suspended in fresh SG Medium and then passed 3 times sequentially at a1:1 ratio onto fresh gelatin-coated wells for 44-48 hr incubationperiods between passages over a total of 6 days. Starting on the 7^(th)day, cells were harvested from all wells as described above, pooledtogether and plated into 1, 9.6 cm² well of mouse embryonic fibroblasts(MEFs) and then maintained and propagated in SG Medium on MEFs aspreviously reported.

Alternatively, 1.2×10⁶ total LB spermatogonia were isolated from theseminiferous tubules of 10 inbred Fischer Rats (F344 NHsd Rats, Harlan,Inc) at 18 days of age, as described above, but with the followingmodifications. In brief, following their isolation, LB spermatogoniawere plated equally into 4, gelatin-coated wells (9.6 cm² wells) of a6-well culture plate. The cells were harvested by gentle pipetting,pelleted at 200×g for 4 min and passed two times sequentially into freshSG Medium at a 1:1 ratio onto fresh gelatin-coated wells for 22-24 hrincubation periods between passages over a total of 2 days. Starting onthe 3^(rd) day, cells were harvested from all wells as described above,pooled together and plated into 1, 9.6 cm² well of MEFs in SG Medium for7 days. The spermatogonia were harvested from off the MEFs by pipettingas described above and incubated in 1×9.6 cm² gelatin-coated well in SGmedium for 6 additional hours to remove any remaining contaminatingsomatic testis cells. After this final selection step on gelatin-coatedplates, the cells were harvested as described above and plated into 1,9.6 cm² well of MEFs and then maintained and propagated in SG Medium onMEFs as previously reported.

In addition to the specific steps described above, details for all cellculture conditions and materials and methods employed for derivingspermatogonial lines from Fischer F344 NHsd rats were performed aspreviously reported for deriving spermatogonial lines from strains ofSprague Dawley rats. See Wu, Z. et al., Biol. Reprod., published on Mar.18, 2009, as DOI:10.1095/biolreprod.108.072645.

Example 4 Restoring Fertility to Rats Deficient in the DAZ-Like (DAZL)Gene

The inventors discovered that, following cryopreservation, long-termcultures of proliferating spermatogonia could be used to restorefertility to rats with a severe form of azoospermia caused by reducedexpression of the germ cell specific RNA binding-protein, DAZL.Additionally, the inventors determined that donor sperm stem cellseffectively restore fertility to DAZL-deficient rats. Accordingly, thisexample illustrates transplantation of spermatogonia to restorefertility in sterile rats.

Animal Care and Use

Protocols for the use of rats in this study were approved by theInstitutional Animal Care and Use Committee (IACUC) at UT-SouthwesternMedical Center in Dallas. Rats used for this study were housed inindividually ventilated, Lab Products 2100 cages in a dedicated roomwith atmosphere controls set to 72° F., 45-50% humidity during a 12 hourlight/dark cycle (i.e. Light cycle=6:00 am-6:00 pm, Central StandardTime adjusted for daylight savings time). Rats were fed Harlan TekladIrradiated 7912, LM-485 Mouse/Rat Diet, 5% fat Diet and a continuoussupply of reverse osmosis water.

Rat Spermatogonial Lines

Seminiferous tubules were isolated from testes of 23-24 day oldhomozygous SD-Tg(ROSA-EGFP)2-4Reh Sprague Dawley rats. Rats of theSD-Tg(ROSA-EGFP)2-4Reh line were produced by pronuclear injection, andexhibit germ cell-specific expression of enhanced green fluorescentprotein (EGFP) throughout male and female gametogenesis, and arereferred to as GCS-EGFP rats. For deriving each spermatogonial line,seminiferous tubules were isolated from an individual rat and thenenzymatically and mechanically dissociated into a cellular suspension togenerate a testis cell culture. The testis cell culture was then used toisolate enriched populations of laminin-binding germ cells, which arehighly enriched in spermatogonial stem cells. The laminin-bindingfraction of germ cells was then used to derive proliferating cultures ofspermatogonia [i.e. rat spermatogonial lines RSGL-GCS9 and RSGL-GCS10]in Spermatogonial Culture Medium (SG Medium). Passage 11, RSGL-GCS9 wasthawed after 396 days of cryo-storage at −196° C. in SG Mediumcontaining 10% DMSO (i.e. Spermatogonial Freezing Medium), and thensub-cultured in fresh SG Medium, for use in experiments between passage13-17.

Germ Cell Transplantation and Colonization

The DAZL-deficient rat line was produced on a Sprague Dawley backgroundand expresses a small hairpin RNA (shRNA) transgene designed tostimulate degradation of transcripts encoding DAZL. Rats in theDAZL-deficient transgenic line are male-sterile due to a defect inspermatogenesis; however, female rats from the same line remained fullyfertile. To prepare recipients for transplantation of spermatogonia,heterozygous DAZL-deficient male rats or their wild-type litter mateswere injected (i.p.) with 12 mg/kg busulfan (4 mg/ml in 50% DMSO) at 12days of age. Busulfan is an alkylating agent cytotoxic to spermatogoniaused to increase the effectiveness of spermatogonial transplantations,and clinically to combat cancer in humans. At 24 days of age (i.e., 12days after busulfan treatment) donor tgGCS-EGFP rat spermatogoniaharvested from culture at either passage 13 (culture day 158) or passage17 (culture day 204) were loaded into injection needles fashioned from100 μl glass capillary tubes at concentrations ranging from 2×10³ to1.5×10⁵ EGFP⁺ cells/50 μl culture media containing 0.04% trypan blue.The entire 50 μl volume was then transferred into the seminiferoustubules of anesthetized rats by retrograde injection through the rete oftheir right testes. The number of EGFP⁺ colonies formed/testis werescored using an Olympus IX70 fluorescence microscope (Olympus, Inc) tovisualize donor cell transgene expression in the seminiferous tubules at30 days following transplantation. Images of recipient testis were takenwith a Nikon SMZ1500 fluorescence stereomicroscope.

Morphometric Analysis of Rat Spermatogenic Cells/Histological Analysisof Rat Testes

Spermatogenesis was evaluated in hematoxylin and eosin (H&E) stained, 3μm thick histological sections prepared from testes of wild-type andDAZL-deficient littermates at 4 months of age, and from DAZL-deficientrecipients at 212 days post-transplantation with spermatogonia (i.e. ˜8months of age). Prior to sectioning, the right testis of each animal wasisolated, incubated overnight at 22-24° C. in Bouin's fixative, washedthoroughly in 70% ethanol and then embedded in paraffin. The averagenumbers of Sertoli cells, gonocytes, type-A spermatogonia (Type-A),intermediate to type-B spermatogonia (Type-B), pre-leptotenespermatocytes (PL), leptotene to early pachytene spermatocytes (L-EP),mid-pachytene to diplotene spermatocytes (MP-D), round spermatids (RS)and elongating spermatids (ES) were scored/tubular cross section.Sections were prepared from triplicate animals for each genotype.Numbers of cells per tubular cross-section were counted for each of theabove categories by morphometric analysis using the Simple PCI software(Simple-PCI, Inc.) in line with an AX70 light microscope (Olympus, Inc).Cells in cross sections of each rat were counted in microscopic fieldsof 15,400 μm² (140 μm×110 μm) from at least 30 tubules/rat, n=3rats/strain. Average counts for each cell type were normalized per 1000Sertoli cells from triplicate rats of each group. Spermatogenic celltypes in wild-type rats were classified based on their morphologies inthe H&E-stained sections and localization to specific stages of aseminiferous epithelial cycle. The spermatogenic stage of each tubuleused to count spermatogonia and Sertoli cells was verified in parallelcross-sections (3 μm) stained by the periodic acid-Schiff method inorder to visualize steps of rat spermiogenesis. In DAZL-deficient rats,spermatogonia were classified as either undifferentiated (Unclip ordifferentiated (Dif) based on their patterns of H&E staining incomparison to wild-type rats. This was due to the inability to clearlyidentify distinct spermatogenic stages in the DAZL-deficient rats.

Histological Analysis of Spermatogonial Types

In wild-type rats, the numbers of undifferentiated and differentiatingspermatogonia per tubular cross section were first scored at stagesVIII, XI, XIII, XIV, II and V of spermatogenesis to avoid scoringdifferentiating spermatogonia in M-phase. Undifferentiated spermatogoniawere clearly distinguished from differentiating types during each stagedue to their relative lack of nuclear staining. As bright field images,undifferentiated spermatogonia were lightly stained throughout thecytoplasm and nucleus, and appeared “pinkish” compared todifferentiating types of spermatogonia. Between stages V and VII,nucleoli A_(a1) spermatogonia became more prominent as theydifferentiated into type A1 spermatogonia. In contrast, types A1 to Bspermatogonia showed increasingly darker staining in distinct regions oftheir chromatin as they differentiated. Types A1 and A2 spermatogoniaoften showed “spots” of darkly stained chromatin situated randomly inthe nucleus, which sharply contrasted from lighter staining chromatinthroughout the rest of the nucleus. Nuclei of types A1 and A2spermatogonia were clearly distinct from nuclei of undifferentiatedspermatogonia. Types A3 to B spermatogonia also showed dark patterns ofchromatin staining with increasing peri-nuclear localization of the mostintensely stained regions.

Fluorometric Analysis of EGFP in Rat Testes

Seminiferous tubules of recipient animals were dissected from the testesof 24 day old rats and then homogenized in 1.5 ml of ice-cold lysisbuffer [50 mM HEPES pH 8.0, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1%TritonX-100, 10 μg/ml aprotinin, 10 μg/ml leupeptin and 1 proteaseinhibitor tablet/12.5 ml] for 30 seconds using a PTA-7 probe on setting5 of a PT10-35 polytron (Kinematica). The homogenates were incubated onice for 15-20 min and then centrifuged at 3000×g for 10 min at 4° C. ina GPR table-top centrifuge (Beckman, Inc.). The supernatant solutionswere centrifuged at 15,800×g for 10 min at 4° C. in a micro-centrifuge(Model 5042, Eppendorf, Inc.), and the resultant supernatant fractionswere then stored at −80° C. Frozen supernatant solutions were thawed onice and then further clarified by centrifugation at 230,000×g, r_(av)(tla-100.3 rotor, TL1000 ultracentrifuge, Beckman, Inc.) for 30 min at4° C. Standards and supernatant solutions from the final centrifugationstep were diluted into assay buffer [100 mM sodium bicarbonate, pH 9.6]and then analyzed for fluorescent intensity using a FL600 fluorescencemicro-titer plate reader (BioTek, Inc.) equipped with filter wheel setsfor maximal excitation at 485 nm and maximal emission at 516 nm.Affinity purified recombinant EGFP with a carboxyl-terminal histidinetag (rEGFP-His) was used as a standard for determining equivalents ofEGFP in lysates prepared from testes of recipient animals. The rEGFP-Hiswas produced by transient expression from the vectorpcDNA6.0-EGFP-V5-His-B following transfection (Fugene 6 transfectionreagent, Roche, Inc.) into COS-7 cells as previously described.

Genotyping Progeny from Recipient Rats

Transgenic rat progeny from crosses between wild-type recipients andwild-type females were genotyped by PCR analysis of genomic DNA usingprimers specific to the EGFP transgene, and the LTR region of thelentiviral transgene used to produce the DAZL-deficient rat line;primers to GAPDH were used to amplify loading controls. Transgene copynumber in F2 progeny from hemizygous crosses between F1 progeny ofrecipients and wildtype females was determined by qtPCR using primers toEGFP and the 18S ribosomal subunit. Genotyping results were verified bySouthern blot hybridization assays of genomic DNA digested with Xmn1 andXba1 using a probe specific for the EGFP transgenes, and for olfactorymarker protein as a loading control. The EGFP probe was isolated as aNhe1/EcoR1 fragment from pLEGFP-C1 (Clonetech, Inc.). The OMP probe wasisolated as a BamH1 fragment from pTOPO-XL:OMP corresponding to basepairs 14268243-14269267 of NW_(—)047561; GI:34857865; RGSC v3.4. EGFPPCR primers: 5′-GTCTCGTGACCCTGACCTACGG-3′ and5′-ATGCCCTTCAGCTCGATGCGG-3; Rat GAPDH PCR primers:5′-ATGATTCTACCCACGGCAAG-3′ and 5′-GCTAAGCAGTTGGTGGTGCA-3′; Lentiviraltransgene PCR primers to LTR region: 5′-AACAGGGACTTGAAAGCG-3′ and5′-ATACTGACGCTCTCGCACCC-3′. Genotyping results were also confirmed inrepresentative progeny by direct visualization of EGFP transgeneexpression in testes and ovaries using a Nikon SMZ1500 fluorescencestereomicroscope.

Inventors morphologically determined that DAZL-deficient rats revealednormal numbers of spermatogonia, but reduced numbers of spermatocytesand round spermatids in their seminiferous tubules, culminating in asevere failure to produce elongating spermatids. Although DAZL-deficientrats fail to produce elongated spermatids, they appear to express anintact spermatogonial stem cell compartment, as evidenced by bothundifferentiated and differentiating populations of spermatogonia withinseminiferous tubules of adult animals. The presence of a functionalspermatogonial stem cell compartment was verified by in vivospermatogenesis colony forming assays in which genetically tagged donorspermatogonia were thawed from cryo-storage, propagated over multiplepassages in culture, and then transplanted into seminiferous tubules ofbusulfan-treated, DAZL-deficient rat testes.

Donor spermatogonial lines were derived from testes of individual GermCell Specific (GCS)-EGFP transgenic rats, which robustly expressed EGFPas a vital marker specifically during all known steps of gametogenesis.The DAZL-deficient recipient rat line also expresses EGFP from itstransgene, but at relatively low levels. By comparison, EGFP is 20-foldmore abundant in testes of GCS-EGFP rats than in DAZL-deficient rats(FIG. 1A). Thus, development of donor spermatogonia from GCS-EGFP ratswas clearly detected following transplantation into DAZL-deficient rattestes, which they colonized 3-fold more efficiently than wildtyperecipient testes (FIG. 1B).

The transplanted spermatogonial stem cells effectively developed intofunctional spermatozoa, which due to the absence of sperm competition,transmitted the donor cell haplotype to progeny >7-fold more efficientlyfrom DAZL-deficient recipients than from wildtype litter mates (FIG.2A-C; Table 1). In each DAZL-deficient recipient, spermatogenesis wasregenerated from the spermatogonial lines that had proliferated inculture (>2 million-fold expansion in cell number) for 5-7 months,yielding 100% germline transmission of the donor haplotype to F1 progenyby natural mating (FIG. 3A-C; Table 1). The GCS-EGFP transgene wasfurther transmitted at Mendelian ratios from F1 to F2 progeny (26%wildtype, 48% heterozygous, 26% homozygous; 81 total pups; n=6 litters)(FIG. 2D). No evidence of tumor formation was observed in any of therecipients or progeny. The regenerative effects of the spermatogoniallines on fertility were also apparent upon histological examination oftestes from DAZL-deficient recipients at 212 days followingtransplantation; 57±6.1% (±SE, n=3) of their seminiferous tubulescontained colonies of spermatogenesis that developed to the elongatingspermatid stage (FIG. 3). This represented a >2000-fold increase in therelative number of elongating spermatids scored/number of Sertoli cellsin transplanted versus non-transplanted DAZL-deficient rat testes (FIG.3).

This is believed the first use of a biochemically tagged, donorspermatogonial line to restore fertility in azoopermic (DAZL-deficient)transgenic rats. Thus, fertility was safely restored to all recipientstransplanted with spermatogonial lines after long-term proliferation ina recently formulated spermatogonial culture medium as described inExample 2 above. Surprisingly, as few as 50,000 donor spermatogoniatransplanted into only a single testis/recipient are able to effectivelyrestore fertility to azoospermic rats with 100% progeny being derivedfrom the cultured cells after thawing from cryopreservation. The donorspermatogonial lines were able to colonize seminiferous tubules ofDAZL-deficient recipients 3-fold more effectively than tubules ofwildtype litter mates, and transmission of the donor cell haplotype bynatural mating was 7-fold greater from DAZL-deficient recipients. Thisdifference in transmission rate by donor versus endogenous germlines foreach recipient model was associated with two apparent factors: 1)greater colonization efficiencies by donor spermatogonia inDAZL-deficient rat testes, and 2) the relative lack of sperm competitionproduced by DAZ-deficient rats.

Example 5 Library of Spermatogonial Stem Cells Created by TransposonMediated Gene Knockout/in Mutants/Clonal Expansion and Selection ofSingle Clone

Inventors successfully applied spermatogonial stem cells to establishtransposon mutagenesis in the rat by taking advantage of the highlyefficient genomic insertion of a transposon (for example SleepingBeauty) equipped with a gene trap cassette. Inventors carried out asmall-scale pilot screen that allowed the isolation of >150 gene trapinsertions after appropriate selection of clonally enrichedspermatogonia in vitro. Transposon insertions were mapped into genes,some of which are implicated in blood pressure, renal function,cholesterol metabolism and other biological processes relevant to humanhealth. Selected spermatogonial stem cell lines were transplanted intothe testes of recipient rats to allow spermatogenesis resulting ingenetically modified spermatozoa in vivo. Recipient males were pairedwith wild-type females for transmission of genetically modifiedspermatogonial genomes directly to rat progeny by natural mating. Thus,by merging the cellular biology of spermatogonia with state-of-the-arttransposon technology, inventors have generated an experimental pipelinefor creating knockout rats using clonally expanded cultures of germlinestem cells.

In order to transmit stem cell genomes through the rat germline,spermatogonial lines containing gene trap Sleeping Beauty insertionswere selected in culture and transplanted to repopulate testes ofsterilized, recipient rats. Testes of DAZL-deficient and wild-typerecipient rats were transplanted with mixed populations ofG418-resistant spermatogonial lines selected as a library estimated tocontain ˜200,000 individual clonal lines with trapped genes (i.e., genetrapping frequency=0.4% transfected cells×15-20% transfectionefficiency×3×10⁶ cells/transfection). Rat progeny from these crosseswere genotyped using PCR primer sets designed to specifically detect theβ-geo cassette within stably integrated Sleeping Beauty, and theGCS-EGFP rat transgene cassette as an inheritable marker for the donorspermatogonial line. No donor-derived pups were detected in litters bornfrom the wild-type female and recipient pair (0/56 pups born; n=6litters) using the G418-selected spermatogonia. However, litters bornfrom a wild-type female and DAZL-deficient recipient pair yielded 100%germline transmission of the GCS-EGFP+ donor cell haplotype (113/113pups born; n=16 litters), wherein the β-geo, Sleeping Beauty marker wastransmitted to 72% of total F1 progeny, averaging 69.2±29.7%GCS-EGFP+/β-geo+ pups/litter (mean±SD; 82/113 pups total born; n=16litters). Thus, the inventors have shown that cultures of spermatogonialstem cells can be genetically modified with Sleeping Beauty transposonsin culture, clonally enriched for gene-trap mutations by selection inG418-containing medium, and then used to produce mutant rats by naturalbreeding. In each rat line, the defined Sleeping Beauty genomic mutationaccurately predicted disruption of gene expression at the transcript andprotein levels.

Plasmids and HeLa Cell Transfections

To generate the gene trap transposon vector, a 4.3-kb SAβ-geopA cassetteconsisting of a splice acceptor (SA—intron/exon2 boundary ofadenovirus), lacZ-neomycin phosphotransferase fusion (β-geo) andpolyadenylation signal from bovine growth hormone gene was cut out ofthe pSAβ-geo vector with XhoI. The ends were blunted with Klenowpolymerase and ligated to SpeI linkers (NEB), after which the SAβ-geopAcassette was cloned into a Sleeping Beauty vector in which the leftinverted repeat of the element had been doubled HeLa cells were seededat a density of ˜3×10⁵ cells per well on 6-well plates 24 hrs prior totransfection. Plasmids containing transposon and transposase weretransfected in ratios 1:1, 10:10, 1:10 and 10:1 (1=50 ng, 10=500 ng).Reactions were filled up to a total of 1 μg with FV4a (CAT) plasmid. Inthe negative control, the transposase containing plasmid was replacedwith the same amount of a Caggs-CRE plasmid. Transfection reagent usedwas JetPEI™-RGD (2 μl JetPEI™-RGD+98 μl 150 mM NaCl mix added to 100 μlplasmid/150 mM NaCl mix per each sample). 48 hrs after the transfectioncells were trypsinized and ⅕ (200 μl of 1 ml suspension) of the cellsfrom each transfected well were re-plated to 10 cm plates and put onG418 selection (0.6 mg/ml). X-gal staining was done using a modifiedprotocol of Sanes et al. (1986). Briefly, cells were washed with PBS,fixed 5 min at RT in 2% formaldehyde plus 0.2% glutaraldehyde in PBS andthen rinsed with PBS and overlaid with the histochemical reactionmixture containing 1 mg/ml X-Gal, 5 mM potassium ferricyanide, 5 mMpotassium ferrocyanide, 2 mM MgCl₂, 0.75 mM HCl and 0.1% Tween-20 in PBSand incubated in 37° C. for 24 hrs.

PCRs

Splinkerette PCR was completed using a Csp6I restriction digest of thegenomic DNA, and LacZ-specific primer pUC3 5′-cga tta agt tgg gta acgcca ggg-3′ and transposon inverted repeat-specific primer T-BalRev5′-ctt gtc atg aat tgt gat aca gtg aat tat aag tg-3′ in addition to thelinker-specific primers. For reverse transcription PCR (RT-PCR), 5 μgDNAseI-treated RNA was reverse-transcribed using the LacZ-specificprimer 5′-ttc tgc ttc atc agc agg ata tcc-3′ and SuperScriptIII(Invitrogen). The single-stranded DNA was PCR-amplified by LacZ-specificprimer LacZ2 5′-acc acg ctc atc gat aat ttc acc-3′ and gene-specificprimer Tbc1d1-P1 5′-atc act tgt gag cac gca ggg aat a-3′, and furtheramplified with nested primers ZRT 5′-gat tga ccg taa tgg gat agg-3′ andTbc1d1-P2 5′-aag gga agg gag ggc atc tag tcc t-3. PCR products werecloned into pGEM-T (Promega) and sequenced. Real-time PCR was used formeasuring relative transcript abundance of trapped genes expressed inrat tissues. Total RNA was extracted from brain and skeletal muscleusing the Trizol reagent (Ambion). The extracted RNA was quantifiedusing the Pico-Green assay (Invitrogen). cDNA was synthesized by priming100 ng total RNA from each tissue using random primers as previouslydescribed. Relative levels of HIP2 and TBC1D1 transcripts in tissues ofeach rat were then measured by qtPCR using SYBR green dye as described.PCR primer sets were designed to specifically amplify HIP2 flankedboundaries of intron 1, 2, 4, 5. PCR primer sets were designed tospecifically amplify TBC1D1 flanked boundaries of intron 1, 2, 3, and 4.

Rat Spermatogonial Stem Cell Lines

Rat spermatogonial lines, RSGL-GCS3 and RSGL-GCS9, was derived usinghighly pure populations of undifferentiated spermatogonia isolated fromthe germ cell specific-EGFP (GCS-EGFP) transgenic line of Sprague Dawleyrats. The GCS-EGFP rat line expresses EGFP specifically during allstages of gametogenesis. Undifferentiated spermatogonia used to deriveRSGL-GCS3 and RSGL-GCS9 were isolated from rat seminiferous tubulesbased on the principle that testicular somatic cells bind tightly toplastic and collagen matrices when cultured in serum-containing medium,whereas spermatogonia and spermatocytes do not bind to plastic orcollagen when cultured in serum-containing medium. The isolatedspermatogonia provide a highly potent and effective source of stem cellsthat have been used to initiate in vitro and in vivo culture studies onspermatogenesis. RSGL-GCS3 was originally derived, sub-cultured andmaintained on feeder layers of irradiated, DR4 mouse embryonicfibroblasts (MEFs) in Shinohara's Medium minus serum and vitamin-A,until frozen in cryo-storage. Upon thawing for the current study,RSGL-GCS3 was propagated on DR4 MEFs using Spermatogonial Culture Medium(SG Medium). RSGL-GCS9 was originally derived, sub-cultured andmaintained on feeder layers of irradiated, DR4 MEFs at 4.5×10⁴ cells/cm²in SG Medium until frozen in cryo-storage, prior to thawing forproduction of mutant rats in the current study, as described below.

Transfection of Rat Spermatogonial Lines with Sleeping Beauty Constructsand Selection of Gene Trap Insertions

Spermatogonial line RSGL-GCS3, was thawed after cryo-storage at passage8 in a spermatogonial freezing medium (SG Freezing Medium) and thenexpanded to passage 10 in fresh SG Medium before harvesting forNucleofection with Sleeping Beauty Plasmids. Spermatogonial lineRSGL-GCS9 was thawed after cryo-storage at passage 9 in SG FreezingMedium and then expanded to passage 11 in fresh SG Medium beforeharvesting for Nucleofection with Sleeping Beauty Plasmids. Theharvested spermatogonia were transfected with DNA test constructs using10 μg total DNA in a suspension of 3×10⁶ spermatogonia/100 μlNucleofection Solution L (Amaxa, Inc.) using settings A020 on theNucleofector (Amaxa, Inc.). For genetic modification of the ratspermatogonial lines with a hyperactive Sleeping Beautytransposon/transposase system, a ˜1:30 ratio of the transposase totransposon plasmids was used for these transfections (i.e., 0.33 μgtransposase:9.67 μg transposon/100 μl Nucleofection Solution L).

Following transfection, the spermatogonia were plated directly into SGMedium at an equivalent of ˜2.5×10⁵ nucleofected cells/9.5 cm² ontofreshly prepared irradiated MEFs. On day 7 following nucleofection,cultures were maintained in SG medium containing 75 μg/ml G418(Invitrogen, Inc.) for an additional 10 days to select for cells thatcontained gene trap insertions. Cultures were fed fresh SG medium withG418 every two days. After this selection period, cultures weremaintained in SG-medium without G418. Following nucleofection, freshMEFs (2×10⁴/cm²) were added into ongoing cultures of spermatogonialcolonies every 10-12 days prior to passaging. At 35-45 days followingnucleofection, individual colonies were picked from cultures using ap200 Eppendorf tip and then the colonies were transferred into wells ofa 96-well plate for Monoclonal expansion of the germlines. In parallel,all of the colonies from the second six well plate from the sametransfection were harvested and pooled together for Polyclonal expansionof rat germlines for an additional 35 days following selection in G418to sufficient numbers for both their cryopreservation (Duplicate vialsat 2×10⁶ cells/line) in liquid nitrogen, and for their transplantationinto recipient rat testes (1.5-3×10⁵ cells/rat), as described below.

Spermatogonial Transplantation

The DAZL-deficient rat line was produced on a Sprague Dawley backgroundand expresses a small hairpin RNA (shRNA) transgene designed tostimulate degradation of transcripts encoding DAZL. Rats in theDAZL-deficient transgenic line are male-sterile due to a defect inspermatogenesis; however, female rats from the same line remained fullyfertile. To prepare recipients for transplantation of spermatogonia,heterozygous DAZL-deficient male rats or their wildtype litter mateswere injected (i.p.) with 12 mg/kg busulfan using our establishedprotocol. At 12 days post-busulfan treatment of the recipients, theG418-resistant donor spermatogonia were harvested from culture andloaded into injection needles at concentrations ranging at 3-6×10⁵spermatogonia/50 μl culture media containing 0.04% trypan blue (Sigma).The entire 50 μl volume was then transferred into the seminiferoustubules of anesthetized rats by retrograde injection through the rete oftheir right and left testis. Rats used for this study were housed inindividually ventilated, Lab Products 2100 cages in a dedicated roomwith atmosphere controls set to 72° F., 45-50% humidity during a 12 hourlight/dark cycle. Rats were fed Harlan Teklad Irradiated 7912, LM-485Mouse/Rat Diet, 5% fat Diet and a continuous supply of reverse osmosiswater. Protocols for the use of rats in this study were approved by theInstitutional Animal Care and Use Committee (IACUC) at UT-SouthwesternMedical Center in Dallas, as certified by the Association for Assessmentand Accreditation of Laboratory Animal Care International (AALAC).

Genotyping Mutant Rat Progeny from Transplanted Recipient Rats

Genotyping primers were specific to the beta-Geo and EGFP transgenes,and the LTR region of the lentiviral transgene used to produce theDAZL-deficient rat line; primers to GAPDH were used to amplify loadingcontrols. Genotyping results were verified by Southern blothybridization assays of genomic DNA digested with XmnI and XbaI using aprobe specific for the EGFP transgenes, and for olfactory marker protein(OMP) as a loading control, and the LacZ portion of the Beta-Geo insertin the Sleeping Beauty transposon construct. The EGFP probe was isolatedas a NheI/EcoRI fragment from pLEGFP-C1 (Clonetech, Inc.). The OMP probewas isolated as a BamHI fragment from pTOPO-XL:OMP corresponding to basepairs 14268243-14269267 of NW_(—)047561; GI:34857865; RGSC v3.4.

Western Blotting

Proteins were extracted from dissected testes and then homogenized in1.5 ml of ice-cold lysis buffer (50 mM HEPES, pH 8.0, 150 mM NaCl, 1 mMEDTA, 10% glycerol, 1% Triton X-100, 10 μg/ml aprotinin, 10 μg/mlleupeptin and 1 protease inhibitor tablet/12.5 ml) for 30 s using aPTA-7 probe on setting 5 of a PT10-35 polytron (Kinematica). Thehomogenates were incubated on ice for 15-20 min and then centrifuged at3000×g for 10 min at 4° C. in a GPR tabletop centrifuge (Beckman, Inc.).The supernatant solutions were centrifuged at 15,800×g for 10 mM at 4°C. in a microcentrifuge (Model#5042, Eppendorf, Inc.), and the resultantsupernatant fractions were then stored at −80° C. Frozen supernatantsolutions were thawed on ice and then further clarified bycentrifugation at 230,000×g, rav (tla-100.3 rotor, TL1000ultracentrifuge, Beckman, Inc.) for 30 min at 4° C. Protein (100 μg perpooled sample per lane) was separated on SDS gels (10-20% acrylamidegradient, Invitrogen, Inc.) and transferred to nitrocellulose membranes.Nonspecific, proteinbinding sites were blocked by incubating membranesovernight at 4° C. in blocking buffer: TBST (Tris-buffered saline withTween-20: 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) containing5% nonfat dry milk. Membranes were washed three times in TBST andincubated for 1 h at 22-24° C. with diluted primary antibody (1/1000HIP2; 1/300 TBC1D1) in blocking buffer. Membranes were washed threetimes in TBST (0.3% Tween-20) and incubated 45 min, 22-24° C. withperoxidase-conjugated, anti-mouse IgG diluted 1:50,000 in blockingbuffer. Membranes were washed three times in TBST and protein bandsdetected using the enhanced chemiluminescence detection method (ECL,Amersham, Inc.).

In summary, the invention is technically less difficult, less expensive,and less time-consuming to implement than conventional technologies inthis field. The invention also guarantees maximal levels of germlinetransmission of mutated genes to rat progeny, by either natural matingor assisted reproduction.

TABLE 1 Progeny from Wildtype and DAZL-Deficient Recipient RatsTransplanted with GCS-EGFP Rat Spermatogonia Days Average Pups Born fromRecipient Passage Days to Gram wt per Cells per to first Number LitterTotal Pups Transplanted Recipient Background Number Analysis R, L testisR, L testis litter Litters Size Born Stem Cells (%)* R946 Wildtype p13212 0.569, 0.341 50,000:0    127 3 11.3 34 3 (8.8) R948 Wildtype p13 2120.735, 0.278 50,000:0    131 3 10 30 11 (36.7) R949 Wildtype p13 2121.054, 0.201 50,000:0    113 3 17.3 52 2 (3.8) Average p13 212 0.786,0.273 50,000:0    123.7 3 12.9 38.7 4.3 (16.4) R942 DAZL-Def p13 2120.572, 0.286 50,000:0    154 3 8.7 26 26 (100) R943 DAZL-Def p13 2120.651, 0.377 50,000:0    141 3 7.7 23 23 (100) R944 DAZL-Def p13 2120.511, 0.326 50,000:0    156 3 8 24 24 (100) Average p13 212 0.578,0.330 50,000:0    150 3 8.1 24.3 24.3 (100) R988 DAZL-Def p17 236 0.523,0.578 150,000:150,000 122 3 12.7 38 38 (100) R989 DAZL-Def p17 2360.489, 0.439 150,000:150,000 128 3 9.3 28 28 (100) R990 DAZL-Def p17 2360.487, 0.371 150,000:150,000 136 3 5.3 16 16 (100) Average p17 2360.499, 0.427 150,000:150,000 128.6 3 9.1 27.3 27.3 (100) Wildtype orDAZL-deficient (DAZL-def) recipient rats were transplanted with either0.5 or 1.5 × 10⁵ EGFP⁺ cells/testis from rat spermatogonial line GCS9 at12 days after busulfan treatment (i.e. 12 mg/kg i.p.) on postnatal day24. At ~75 days post-transplantation recipients were paired with 75-80day old wildtype female rats. Spermatogonia line GCS9 was harvested frompassages number 13 and 17, which corresponded to 158 and 204 days inculture, respectively, prior to their transplantation. RecipientsR942-R949 were littermates born from a hemizygous, transgenicDAZL-deficient female and a wildtype Sprague Dawley male. No progenywere born from breeder pairs of un-transplanted, busulfan-treatedDAZL-deficient males and wild-type females (n = 3 breeder pairs).Breeder pairs of untreated, wild-type male litter mates ofDAZL-deficient rats and wild-type female rats from Harlan, Inc. produced15.5 ± 4.5 pups/litter (+/−SEM, n = 8 litters from 3 breeder pairs).*Percent GCS-EGFP⁺ F1 progeny.

TABLE 2 Components of Rat Spermatogonial Culture Media SA Medium*SR-LE-Medium** SG Medium** RSFM*** B27-Vitamin A (concentration)(concentration) (concentration) (concentration) Supplement **** StemProbasic (1x) StemPro basic (1x) DMEM: HamsF12 (1x) MEM α (1x) d-BiotinAnti-biotic/mycotic (1x) Anti-biotic/mycotic (1x) Anti-biotic/mycotic(1x) Penicillin (50 units/ml) BSA, fatty acid free Fraction VL-glutamine (2 mM) L-glutamine (2 mM) L-glutamine (6 mM)^(†)Streptomycin (50 μg/ml) Catalase 2-mercaptoethanol (50 μM)2-mercaptoethanol (50 μM) 2-mercaptoethanol (100 μM) I-glutamine (2 mM)L-Carnitine HCl Glucose (6 mg/ml) Glucose (6 mg/ml) B27-vitamin A Supp.(1x) 2-mercaptoethanol (100 μM) Corticosterone MEM vitamin (1x) MEMvitamin (1x) Rat GDNF (20 ng/ml) Hepes (10 mM) Ethanolamine HCl NEAA(1x) NEAA (1x) Human bFGF (20 ng/ml) palmitic acid (4.8 μM) D-Galactose(Anhyd.) Estradiol (30 ng/ml) Estradiol (30 ng/ml) palmitoleic acid(0.42 μM) Glutathione (Reduced) Pyruvic Acid (30 μg/ml) Pyruvic Acid (30μg/ml) stearic acid (1.76 μM) Insulin (Human, recombinant) Lactic Acid(1 μl/ml) Lactic Acid (1 μl/ml) oleic acid (2.0 μM) Linoleic AcidAscoribic Acid (100 μM) Ascoribic Acid (100 μM) linoleic acid (5.4 μM)Linolenic Acid B27-vitamin A Supp. (1x) B27-vitamin A Supp. (1x)linoleic acid (0.85 μM) Progesterone Rat GDNF (10 ng/ml) Rat GDNF (10ng/ml) GFR α 1 (300 ng/ml) Putrescine•2HCl Human bFGF (10 ng/ml) HumanbFGF (10 ng/ml) Murine LIF (1000 units/ml) Sodium Selenite (1000X)StemPro Supplement (1x) StemPro Supplement (1x) Rat GDNF (40 ng/ml)Superoxide Dismutase Mouse EGF (20 ng/ml) Human bFGF (1 ng/ml)T-3/Albumin Complex Murine LIF (1000 units/ml) Insulin (25 μg/ml) DLAlpha-Tocopherol Insulin (25 μg/ml) Apo-transferrin (100 μ/ml) DL AlphaTocopherol Acetate Biotin (10 μg/ml) Putrescine (120 μM) Transferrin(Human, Iron- Poor) Progesterone (60 ng/ml) Na₂SeO₃ (60 nM)Apo-transferrin (100 μg/ml) BSA (6 mg/ml) Putrescine (60 μM) Na₂SeO₃ (30nM) BSA (5 mg/ml)

TABLE 3 Vendor and Catalog numbers for SA and SG media componentsComponent Supplier, Catalog Number 1. Base Media/Nutrient Supplements:StemPro34 SFM Base Invitrogen, 10639-011 40x StemPro34 nutrientsupplement Invitrogen, 10639-011 Dulbecco's Modified Eagle's Sigma,D8437 Medium:HamsF12 nutrient mix (1:1)* B-27 Supplement minus vitaminA* Invitrogen, 12587-010 2. Added Small Molecule/Nutrient Components:d-(+) Glucose Sigma, G7021 L-glutamine* Invitrogen, 25030-149 50xAntibiotic-Antimycotic* Invitrogen, 15240-062 50x MEM VitaminsInvitrogen, 11120-052 50x Non-essential Amino Acids Invitrogen,11140-050 d-Biotin Sigma, B4639 Pyruvic Acid, Sodium Salt Sigma, P4562dl-Lactic Acid (60% Solution) Sigma, L7900 Ascorbic Acid Sigma, A4034Sodium Selenite Sigma, S5261 Putrescine Sigma, P5780 Progesterone Sigma,P8783 β-Estradiol 17-cypionate Sigma, E8004 2-Mercaptoethanol* Sigma,M3148 3. Added Polypeptide Components: Bovine Apo-transferrin Sigma,T1428 Bovine Serum Albumin Calbiochem, 126609 Bovine Insulin Sigma,11882 ESGRO (mouse LIF) Chemicon, ESG1107 Recombinant mouse EGF Sigma,E4127 Recombinant human basic FGF* Sigma, F0291 Recombinant rat GDNF*R&D Systems, 512-GF-050

What is claimed is:
 1. A composition comprising an isolated ratspermatogonial stem cell and spermatogonial stem cells expandedtherefrom and a culture medium, wherein the culture medium comprises:1:1 ratio of Dulbecco's Modified Eagle Medium (DMEM) to Ham's F12nutrient mixture, from about 10 ng/ml to about 30 ng/ml glialcell-derived neurotrophic factor (GDNF), from about 10 ng/ml to about 30ng/ml Fibroblast Growth Factor-2 (FGF2), from about 50 to about 120 μM2-mercaptoethanol, from about 3 mM to about 10 mM L-glutamine, and a 1×concentration of B27 minus vitamin A supplement solution.
 2. Thecomposition of claim 1, wherein the isolated stem cell is not DAZ-like(DAZL) deficient.
 3. The composition of claim 1, wherein the isolatedstem cell comprises SEQ ID NO:5 and SEQ ID NO:6.
 4. The composition ofclaim 1, wherein the isolated stem cell is obtained from stem cellscultured from about 30 days to about
 150. 5. The composition of claim 1,wherein the isolated rat spermatogonial stem cell is in culture betweenabout 158 and about 204 days.
 6. The composition of claim 1, wherein theisolated rat spermatogonial stem cell has a doubling time of betweenabout 8 and about 9 days.
 7. The composition of claim 1, wherein theisolated rat spermatogonial stem cell has a doubling time of no lessthan 8.4 days.
 8. The composition of claim 1, wherein the isolated ratspermatogonial stem cell expands no less than about 20,000 times ascompared to the number of cells seeded in culture.
 9. The composition ofclaim 1, wherein the composition is frozen.
 10. The composition of claim1, wherein the isolated rat spermatogonial stem cell comprises one ormore genetic mutations.
 11. The composition of claim 10, wherein theisolated rat spermatogonial stem cell further comprises a nucleotidesequence flanking the one or more sites of the one or more geneticmutations comprising SEQ ID NO:5 and SEQ ID NO:6.
 12. The composition ofclaim 1, wherein the isolated rat spermatogonial stem cell furthercomprises a nucleotide sequence encoding one or more heterologous aminoacid sequences.
 13. The composition of claim 12, wherein the one or moreheterologous amino acid sequences is green florescent protein.
 14. Thecomposition of claim 1, wherein the isolated rat spermatogonial stemcell is resistant to G418.
 15. The composition of claim 1, wherein thecomposition is free of somatic testis cells.
 16. The composition ofclaim 1, wherein the composition further comprises one or a combinationof the following: (i) a nucleic acid molecule comprising a nucleic acidsequence encoding a transposon or a fragment thereof; and (ii) a nucleicacid molecule comprising a nucleic acid sequence encoding a transposase.17. A method for culturing isolated rat spermatogonial stem cellsisolated from testis cells according to claim 1 comprising: allowing theisolated rat spermatogonial cells to adhere to a surface in a culturevessel; and culturing the isolated rat spermatogonial stem cells inculture medium according to claim 1.